9

RNA biosynthesis

Gene expression involves a sequential flow fluence the frequency with which any
of information. In the first stage of this transcription unit is transcribed (they are to
process, known as transcription, one strand be fully described in forthcoming sections).
of double-stranded DNA is copied into an These elements may include nucleotide
RNA. In contrast to DNA replication, the sequences that are recognized by RNA
genome is not copied in its entirety. Rather, polymerase or some other DNA-binding
defined units of genetic information are protein that modulates RNA synthesis.
copied into RNA molecules which may A given recognition sequence, or one like
function as messenger (mRNA), form a it, may thus be found associated with all
component of ribosomes (rRNA) or have an the genes influenced by a particular pro-
adapter function ( tRN A). All three of these tein. Such sequences are known as con-
RNA products take part in the subsequent sensus sequences. Typically, they are not
stage of gene expression, known as trans- totally conserved but are a consensus from
lation, in which mRNAs are used as coded which any similar sequence deviates only
messages for the synthesis of protein. marginally.
Translation is the subject of chapter 12. Associated with the controlled transcrip-
tion of a gene, there may also be elements,
such as regulator genes, that are indepen-
9.1 CONVENTIONS AND TERMS dently transcribed and cause the formation
ASSOCIATED WITH RNA of proteins that influence the frequency
BIOSYNTHESIS of transcription. Segments of DNA that
influence the activity of genes with which
RNA biosynthesis is called transcription. they are contiguous are described as cis-
A transcription unit is a segment of DNA acting. This differentiates them from trans-
(gene or genes) that is transcribed (copied) acting elements that give rise to diffusible
into a single RNA molecule by the enzyme products that can act at many sites whether
RNA polymerase. Associated with this on the same or different chromosomes.
segment of the genome are various non- Trans-acting elements are transcribed and
transcribed DNA elements such as pro- translated into proteins called trans-acting
moters, enhancers and operators that in- factors or transcription factors. These

R. L. P. Adams et al., The Biochemistry of the Nucleic Acids

© Roger L. P. Adams, John T. Knowler and David P. Leader 1992
340 RNA biosynthesis

influence transcription by interacting with is the bacterial enzymes, particularly that of

promoters, enhancers and other cis-acting E. coli, that are most fully characterized
elements. (reviewed in [2-4]). The complete E. coli
RNA is synthesized in a 5' ~ 3' direction enzyme or holoenzyme, of Mr 449068,
from the complementary strand of the contains five polypeptide chains: two
DNA. However, when describing the a-chains of Mr 36 512; one P-chain of Mr
nucleotide sequence of genes it is conven- 150619; one P'-chain of Mr 155162 and
tional to describe the coding strand as that one a-chain of Mr 70 236.
having the same sequence as the RNA The P and P'-subunits each contain a
transcript (except that in the RNA, uridines Zn 2 + ion [5] which is essential for catalytic
replace thymidines). The point at which activity. The enzyme requires all four
transcription starts is called the initiation ribonucleoside triphosphates and a divalent
site or start site. Sequences conventionally metal ion which in vivo is Mg2 + but in vitro
written to the left of the transcription unit can be Mn2+. When isolated, the enzyme
are described as being 5' to the start site. normally consists of a mixture of the holo-
Such elements are also described as being enzyme and the core enzyme from which
upstream whereas those on the 3' -side of the a-subunit, commonly known as the
the start site are described as downstream. sigma factor, is missing. The core enzyme
Individual nucleotides are numbered away is the catalytic component of the enzyme.
from the start site and are given positive It has a general ability to bind to DNA
values when downstream and negative and, if the DNA is nicked, can catalyse the
values when upstream. Thus, a nucleotide synthesis of RNA from the nicks. It is not,
20 base pairs before the start site is -20 and however, able to catalyse specific initiation.
one 10 base pairs after the start site is at The addition of a sigma factor to the core
position +10. enzyme reconstitutes a fully active holo-
enzyme [6] that now has reduced affinity for
non-specific DNA sequences but a con-
9.2 TRANSCRIPTION IS BEST siderably increased affinity for specific
UNDERSTOOD IN PROKARYOTES recognition sites near the beginnings of the
sequences to be transcribed. These se-
9.2.1 E. coli RNA polymerase quences are known as promoters. Their
nature and role in the initiation of tran-
The enzymes that catalyse the synthesis of scription by RNA polymerase is further
RNA from a DNA template are known as discussed in the following section. The
DNA-dependent RNA polymerases. These transcription of most genes requires the
enzymes require ribonucleoside triphos- predominant or primary sigma factor. In
phates as substrates and transfer nucleoside many species, however, there are alternative
monophosphates onto the 3'-0H terminus sigma factors that recognize the promoters
of the growing RNA chain. Polymerization of different sets of genes. Alternative RNA
is thus in a 5' ~ 3' direction and produces polymerase sigma factors are thus a means
an RNA chain the sequence of which is for the selective expression of specific genes
determined by Watson and Crick base- and this is considered in section 10.3.1.
pairing with the DNA template. RNA The function and mode of action of
polymerase activity was first detected in rat the subunits within the core enzyme are
liver nuclei by Weiss and Gladstone [1] but it incompletely understood. Numerous studies
Transcription in prokaryotes 341

employing inhibitors, affinity labelling, helix is unwound. Finally, RNA synthesis

reconstitution and genetic methods [7 -9] is initiated (reviewed in [10]).
confirm that a, P and P' are all necessary
components of the core enzyme. They also (a) Binding of RNA polymerase to
show that the P' -subunit appears to be prokaryotic promoters
largely responsible for DNA binding. It is
basic as would be expected of a protein with The binding of RNA polymerase to tran-
such a function and it binds the polyanion scriptional promoters appears to require an
heparin that inhibits transcription in vitro. initial non-specific association with DNA.
The psubunit has been implicated in binding There are calculated to be 4 x 106 of these
both the substrates and the products of non-specific binding sites in the E. coli
catalysis and it contains at least a portion of genome and the fact that this corresponds
the catalytic site for RNA synthesis. It is also closely to the total number of base pairs
the site of action of antibiotics such as the (4.5 x 106) emphasizes the non-specific
streptolydigins which inhibit RNA chain nature of the interaction. The non-specific
elongation and rifamycins which inhibit binding constant of approximately 2 X
initiation by preventing the formation of the lOll M- 1 is strong when set against the bind-
first phosphodiester bond. ing of many proteins to their ligands. Never-
There are approximately 7000 RNA theless, it is relatively weak compared with
polymerase molecules in an E. coli cell and, the binding to specific promoters and the
depending on the rate of growth, about DNA remains in the double-stranded
2000-5000 of these will ·be actively tran- (closed) form.
scribing RNA at any given time. The RNA The precise role of non-specific binding
polymerases of other bacteria, as well as and the mechanism by which the enzyme
those of blue-green algae, are similar to moves from them to specific promoters is
those of E. coli and contain subunits that are not fully understood. Von Hippe! and
homologous to the a, p, P' and a-chains. coworkers [11, 12] have shown that three-
Indeed, in many though not all cases, the dimensional diffusion processes are not
homology is such that reconstituted enzymes fast enough to account for the transfer.
containing heterologous mixtures of sub- Furthermore, they have reviewed evidence
units derived from different species are still that suggests that non-specific binding is
catalytically active [3]. electrostatic and that, in this mode, facili-
tated transfer may occur by sliding along the
DNA [13, 14] or by hopping [15] or a com-
9.2.2 Initiation of prokaryotic bination of both processes [16]. Heumann
transcription and coworkers [17, 18] have presented data
favouring facilitated diffusion and have
The mechanism by which RNA polymerase described the non-specific DNA as an
initiates transcription has been most exten- 'antenna' along which RNA polymerase
sively studied with the enzyme from E. coli moves to the promoter. They reasoned
and has been shown to occur in three stages. that if such an effect occurred, occupancy of
First, the holoenzyme recognizes and binds the promoter should be positively influenced
to specific promoters. Second, this binding by an increasing length of flanking sequence.
leads to the formation of the so-called 'open Such an effect was demonstrated with
complex' in which a portion of the double downstream but not upstream sequences.
342 RNA biosynthesis

a ~cTTGACa~ ~ ~ ~g TMAaT ca~

-50 -40 -30 -20 -10 +1 +10

Fig. 9.1 Consensus sequence for the E. coli promoter.

Transfer from non-specific binding to represent weakly conserved nucleotides and

a tight binding complex with a specific no letters are inserted where there is no
promoter depends on the presence of sigma evidence for conservation. The importance
factor. The binding constant for the asso- of the consensus sequences has also been
ciation varies from 10 12 to 10 14 M- 1 and the investigated in promoter mutants [21, 22]
strength of the complex, referred to as which are described as down or up mutants
promoter strength, is strongly related to the depending on whether they decrease or
efficiency with which the associated gene is increase transcription from the promoter.
transcribed. In an analysis of 98 E. coli mutants, nearly
E. coli RNA polymerase holoenzyme will all followed a general rule that down mu-
protect promoter regions from nuclease tants showed decreased homology with the
attack. The length of the protected segment consensus sequence whereas up mutants had
varies with the nuclease and digestion increased homology [22]. The length of
conditions used but it is clear that the DNA between the -10 and -35 regions is
enzyme binds asymmetrically to the start also fairly stable; 100 of the 112 promoter
site of transcription such that it covers up sequences compiled by Hawley and McClure
to 50 nucleotides on the 5' -side of the gene [21] had 17± 1 intervening nucleotides,
and up to 20 nucleotides into the gene [13]. however sequences ranging from 15 to
Within the 5' -side of this protected area, 20 nucleotides are known. Similarly the
there are two segments, centred around distance between the -10 region and the
nucleotides -10 and -35, the sequences of transcription start site can vary from 5 to
which are strongly conserved. It is these 9 nucleotides.
segments that are the promoters to which An overall picture of E. coli promoters is
the holoenzyme binds. The -10 sequence, thus of fairly tightly constrained sequence
often known as the Pribnow Box, has the and spatially defined elements but never-
consensus sequence TATAAT and the -35 theless with a substantial degree of vari-
region has a consensus sequence TTGACA ·ability. Many promoters are in fact weak;
[19, 20]. It should be emphasized that their function relies on and is controlled by
not all promoters have these sequences. other proteins that recognize regulatory
However, any given promoter will vary elements and assist initiation (These con-
from the consensus at no more than a few trolling factors are considered in chapter
positions and this variation is more likely to 10.) Notwithstanding this, there are still
occur in some nucleotides than in others. problems. First, it is not clear that there is
Thus, in a compilation of 112 well-defined sufficient information in what is understood
E. coli promoter regions [21], a consensus of promoter elements to define their un-
sequence for the region -50 to +10 was ambiguous recognition by RNA polymerase.
derived. This is illustrated in Fig. 9.1, Second, there is little correlation between
where upper case letters indicate strongly initial promoter recognition (rate of complex
conserved nucleotides, lower case letters formation) and promoter strength defined
Transcription in prokaryotes 343

-35 -16 -10 +1

t + +
• • •TTGACA • • • • • • • • • • • • • • • • TATAAT • • • ••• • • •
(a)

(b)

Fig. 9.2 Contact points for E. coli RNA polymerase on the promoter of the P-galactosidase gene. The
sequence of the promoter coding strand (a) is aligned with the distribution of contact points (darkened
areas) on one face of the DNA double helix (b).

as rate of RNA synthesis [23]. O'Neil [24], hydrogen-bond pattern [12], may be im-
in an analysis of 52 well-characterized portant in enzyme recognition. That three-
promoters, found that on average they dimensional structure must be important is
conserved only 3.9 bases in the -35 con- indicated by the finding that the length of
sensus sequence and 4.2 bases in the -10 the DNA segment between the -10 and
consensus sequence. This level of degener- -35 regions has considerable influence on
acy could lead on average to promoter promoter strength. In the B form of DNA,
recognition every 200 base pairs of DNA; the -35, -10 and -16 regions all lie on the
obviously with many false positives. Clearly, face to which RNA polymerase binds; a
additional information must specify an RNA change in the length of the intervening
polymerase recognition site. segment would skew this arrangement [25]
Some further information comes from the which might also be influenced by super-
analysis of contact points between RNA coiling [26]. One recent suggestion is that
polymerase and its promoter. Such experi- there are three classes of promoter de-
ments are performed by examining the pending on whether there are 16, 17 or
extent to which the polymerase can protect 18 nucleotides between the -10 and -35
specific nucleotides from base-modifying regions [24].
reagents. Alternatively, mutations and Experiments with mutant sigma factors
base-specific reagents can be tested for their and with mutant promoters demonstrate
effect on enzyme binding. Contact points that the factor recognizes both the -10 and
are heavily concentrated in the -35 and the, -35 regions on the promoter ([27] and
-10 consensus regions of the DNA and are references therein). Two out of four highly
also clustered in the -16 area [20]. In all conserved regions of the polypeptide are
three areas, the contact nucleotides are involved in the recognition.
not always those that are most strongly The promoters of bacteriophage that
conserved (Fig. 9.2) so it would appear rely on the RNA polymerase of their host
that features additional to base sequence, compete very effectively for the enzyme.
such as three-dimensional structure and It is perhaps surprising, therefore, that the
344 RNA biosynthesis

consensus sequence of 29 bacteriophage T4 R +p fast ) RPc rate-limiting step RPi

promoters differs significantly from that of
E. coli [28]. The difference raises the poss- major induced-fit
ibility that additional proteins are involved RPo
in their recognition. Variant prokaryotic R is RNA polymerase and P is the promoter.
RNA polymerase promoters associated RPc is the closed enzyme promoter complex
with the expression of complex gene sets in which the DNA is still double-stranded
are discussed in section 10.3.1. and the -10 and -35 regions are not fully
registered with the enzyme. RPi is envisaged
as an intermediate stage in which the DNA is
(b) Formation of an open promoter complex
still double-stranded but the -10 and -35
and the initiation of RNA synthesis
regions are in register and the enzyme is
strictly positioned with respect to the DNA
With the tight binding of RNA polymerase backbone. During the transition between
to a promoter site, there is a rapid transition RPc and RPi, the DNA is topologically un-
to the so-called open-complex in which the wound and the unwinding (untwisting or
DNA is partially unwound (Fig. 9.3). negative writhing) is converted into strand
Unwound regions of DNA are accessible separation in the final step; the formation of
to base-modifying reagents. Siebenlist et al. the open complex, RP 0 .
[20] exploited this and used dimethyl sul- Transcription in prokaryotes is most
phate to methylate the N1 and N3 positions commonly initiated with a purine nucleotide
of adenines and cytosines, respectively, in at a site 6 or 7 bp downstream from the -10
the unwound segments of the open complex. region. Of 88 promoter start sites examined
They showed that a 12 bp region of the by Hawley and McClure [21], A and G
promoter is unwound and stretches from occurred at position + 1 of the coding strand
the middle of the -10 region to just beyond 45 and 37 times, respectively, and pyrim-
the RNA start site (specifically from -9 to idines occurred in only six sequences. They
+ 3). The fact that this region overlaps with also demonstrated that pyrimidines were
a contact point between one strand of the preferred at positions -1 and + 2 with CAT
DNA and sigma factor [27] indicates that as the overall consensus sequence for the
the essential role of this subunit in correct triplet -1, + 1, +2 (Fig. 9.1). Thus, a con-
initiation may be associated with the form- sensus transcript would begin pppApU.
ation of open complexes. Buc and colleagues The binding of the initiation nucleotide to
[29-32] have investigated both the opening RNA polymerase is an order of magnitude
and the associated unwinding of strong and stronger than that of succeeding nucleotides
weak promoters. They find that although and it has been suggested that it occupies a
the kinetics of opening are different, both separate initiating site on the enzyme [33].
open to the same extent, i.e. about 12 bp. At an early point in the incorporation of
They have further investigated the role of nucleoside phosphates into new transcripts,
supercoiling in the process and have sug- two further changes occur in the initiation
gested that initially the DNA is wrapped complex. In the first of these, the open com-
around the polymerase in a nucleosome-like plex is transformed into a ternary complex
form [32]. In at least some promoters, the which is much more resistant to dissociation
open complex is then believed to form via by high salt concentrations, is resistant to
an intermediate step: inhibition by rifampicin and in which RNA
 ~ Holoenzyme binds to promotor

(a) Closed complex

+Partial DNA unwinding

(b) Open complex

t Incorporation of first two nucleotides

G . ..----1
.
( c ) Ternary complex

Release of sigma and continuation of transcription

Direction of synthesis
(d) Elongation complex
s' 3'

Fig. 9.3 Diagrammatic representation of the stages in the initiation of transcription. On the elonga-
tion complex (d) 1, 2 and 3 represent the active centres for the removal of positive supercoiling, the
removal of negative supercoiling and elongation, respectively.
346 RNA biosynthesis

DNA

RNA {
produc~ 0

Incoming ~riphosphate

Direc~ion of syn~hesis
s' 3'

Fig. 9.4 A diagrammatic representation of the biosynthesis of RNA from the strand of the DNA that
acts as a template.

polymerase is more resistant to denaturation 9.2.3 Elongation of prokaryotic

and to proteolytic enzymes. DNase foot- transcripts
printing (section A.9.2), photoaffinity
labelling and rapid kinetics have been used Elongation proceeds by the successive
to study the structure of this ternary complex addition of ribonucleoside monophosphates
[34-36]. They show that RNA polymerase from substrate triphosphates on to the
remains more or less fixed on the promoter 3' -OH terminus of the growing RNA chain
as the first eight nucleotides are polymerized (Fig. 9.4). The nature of the incoming
into RNA. At this time, sigma factor is still nucleotide is governed by Watson-Crick
attached to the enzyme and the promoter base-pairing rules and bond formation is
shows increased sensitivity to DNase at accompanied by the release of pyrophos-
position -25, perhaps indicating bending phate. The reaction can thus be represented
of the DNA in this region [34]. Sigma factor as:
is released after the polymerization of 10 nZTP
nucleotides [34] and the release is apparently
pppXpY pppXpY (pZ)11 + nPfl
dependent on and driven by the nucleotide X, Y and Z can be any of the four ribo-
sequence of the promoter [35]. Only after nucleotides although evidence has already
the incorporation of 10 nucleotides is there been presented for the preferred insertion
movement of the enzyme from the promoter of purines as the 5 '-nucleotide. The elonga-
and a commitment to elongation [34]. The tion complex is diagrammatically illustrated
released subunit is recycled and continued in Fig. 9.3(d).
elongation of the transcript is catalysed by There is a mechanical problem posed by
the core enzyme (Fig. 9.3). the progress of RNA polymerase that is best
Transcription in prokaryotes 347

conceptualized by a simple model. Take a natural template at 12-19 nucleotides

a double stand of fixed cord, such as the per second [41]. This is slower than the
double string employed to draw a blind. If theoretical rate of up to 60 nucleotides per
this is rotated repeatedly clockwise, a double second because transcription from natural
helix is produced that can serve as our model templates is not uniform. Many groups have
of DNA. Now insert a pencil through the shown that elongation is a discontinuous
helix and move it away from you between process in that there are pause sites along
the strings. You have, in effect, a mechanistic the DNA at which RNA polymerase slows
model of RNA polymerase moving along or stops. Elongation is rapid between these
the DNA helix and, as you move the pencil, sites (e.g. [42]). Pausing occurs at GC-rich
the string will overwind in front of the pencil regions and at points 16-20 nucleotides
and underwind behind. This is exactly what downstream of regions of dyad symmetry.
is considered to happen in transcription. At these latter regions the delay is associated
The DNA will become positively supercoiled with the formation, by the nascent RNA, of
in front of RNA polymerase and negatively stem-loop structures either because it base
supercoiled behind it [37]. If we now con- pairs with itself or with the coding strand of
tinue our experiment, we rapidly reach a the DNA [43, 44]. Pausing is a necessary
point at which we cannot push the pencil preliminary to termination (section 9.2.4)
any further; there is too much resistance and premature termination can occur at
from the overwound string and it cannot be internal pausing sites (section 10.2). Viral
overwound anymore. In a cell, such over- RNA polymerases are apparently not
winding must be released. One way to do delayed by the DNA of their hosts and
this would be to allow the DNA and tran- have been reported to elongate at up to
scribing machinery to rotate around each 200 nucleotides per second [45].
other. This immediately appears unlikely. A number of inhibitors have proved
The enormously long filament of DNA useful for the study of elongation [41-45].
could hardly rotate and the concept of the The antibiotics rifampicin and streptovaricin
growing nascent RNA chain rotating around bind to the p subunit and block initiation
the DNA is almost as bizarre. The solution whereas streptolydigin also binds to the
proposed by Liu and Wang [37], in their p subunit but interferes with the elongation
twin-supercoiled-domain model, was that steps. Another agent which prevents elong-
waves of supercoiling generated by the ation is actinomycin D. However, it does
transcribing polymerase would be released so, not by binding to the enzyme, but by
enzymically. DNA gyrase (topoisomerase complexing with deoxyguanosine residues
II) would remove the positive supercoiling in the DNA template and thus preventing
ahead of the RNA polymerase and DNA movement of the core enzyme along the
topoisomerase I would remove negative template (section 7.2.3).
supercoiling behind it. Wang and coworkers
[38, 39] have since used inhibitors of the
different topoisomerases to confirm the 9.2.4 Termination of prokaryotic
existence of waves of supercoiling in vivo. transcripts
Studies in vitro [40] have also provided
supporting data. Three events are required when the tran-
RNA polymerases from a wide variety of scription of a gene is terminated; elongation
bacteria catalyse the synthesis of RNA from ceases, the transcript is released and RNA
348 RNA biosynthesis

polymerase is released. Studies with pro- a minimum of 6 bp, strengthen the efficiency
karyotes have demonstrated that these of termination as does the incorporation
events occur by at least two mechanisms into the transcript of nucleotide analogues,
that are known as factor-independent such as bromo- or iodo-CMP, that stabilize
termination and factor-dependent ter- G:C pairs. Conversely, weakening of G:C
mination. Both of these have been the pairs, such as occurs when inosine is sub-
subject of reviews [29, 46-48]. Before stituted for guanosine, decreases termination
discussing each in turn it should be stressed efficiency [46].
that the precise identification of termination Shortly after the GC-rich region, a run of
sites is not easy. The 5' -end of a prokaryotic four to eight adenylates in the template will
transcript can be unambiguously identified dictate that a string of uridylate residues
by its triphosphate group. No such marker are transcribed into the nascent RNA.
defines the terminus of transcripti~n and an Decreasing the number of these residues
observed 3' -end may be the result of post- has been shown to reduce the efficiency
transcriptional cleavage. of termination [50] and it appears that
Termination has largely been studied the importance of the sequence resides
in vitro using methods that involve the in the very unstable nature of the rU :dA
synthesis, by purified RNA polymerase, of hybrid [51]. Thus three features, the dis-
specific transcripts from bacteriophage or ruption of the RNA:DNA hybrid caused
from defined bacterial genes. Commonly by G:C base-pairing, the pausing of RNA
used systems include the bacterial trp polymerase and instability of the rU :dA
operon (genes encoding the enzymes that region, all combine to facilitate the release
synthesize tryptophan), the DNA coliphages of the transcript from the template. O'Hare
T7, T3, ¢X174 and bacteriophage lambda. and Hayward [52], working with a termin-
Termination occurs at defined sites in these ation site, T1, of coliphage T7, found that
systems and, in a number of them, concern in vitro recognition of the stop signal and
that the sites might be artifacts of in vitro release of RNA occurred with a t 112 of 3 min
incubation conditions has been dispelled by whereas release of RNA polymerase took
confirmation of their use in vivo. 12 min. However, more recent analyses
indicate that the enzyme is released within
(a) Factor-independent termination 13 s of the cessation of elongation [53]. Platt
[271] has suggested that RNA polymerase
Sites on the DNA at which independent undergoes a conformational change to a
termination occurs have a characteristic termination complex at some time in this
structure that comprises a GC-rich inverted process and the concept is supported by the
repeat followed, on the template strand, isolation of polymerase mutants that are
by a run of adenylate residues. The former modified in termination efficiency. Arndt
of these regions results in the formation of and Chamberlin [54] have suggested that the
GC-rich regions in the transcript, which are RNA hairpins bring about this conform-
able to base-pair into a 'hairpin' or stem- ational change by converting the ternary
loop structure (Fig. 9.5). Such loops, which elongation complex into what they call a
typically contain seven to ten G:C base 'release mode'.
pairs, have been shown to cause RNA Some recent data [55, 56] suggest that
polymerase to pause [49]. Mutations that the above picture of factor-independent
lengthen the region of dyad symmetry, over termination may be somewhat simplistic. It
Transcription in prokaryotes 349

(a)

/)oc
--...
""~~~~~'\),
. . uuu' "\
_,, """ ' G-cU U unstable bonds
C-G
C-G
C-G
G-c
,c-G
I \
\ I
(b)
'-~
Fig. 9.5 Independent termination. RNA polymerase is omitted from the diagram for reasons of clarity
(adapted from Platt [271]). (a) point at which elongation stops. (b) formation of hairpin followed by
termination.

was demonstrated that sequences associated structure. It recognizes and binds to the
with the promoter can have a profound nascent RNA transcript and the RNA-
influence on termination but the way m binding domain is in the first 151 amino
which this occurs is totally unknown. acids of the subunit [58]. It binds to 13±1
nucleotides per monomer and 78±6 nucleo-
(b) Factor-dependent termination tides per hexamer [59] such that the poly-
nucleotide appears to be wrapped around or
Roberts [57] discovered a protein, which he condensed within the protein oligomer [60].
called rho (p), that when added to an E. coli The protein also exhibits RNA-dependent
transcription system in vitro causes the A TPase activity that is located in the car-
generation of transcripts with discrete boxyl two thirds of the polypeptide [61]
3'-ends. The protein is basic in character and is required for the helicase activity that
and in solution consists of six identical, brings about the unwinding of RNA:DNA
419 amino acid subunits arranged in a ring hybrids [62-64].
350 RNA biosynthesis

As with independent termination, the antitermination of E. coli rRNA synthesis

activity of rho requires RNA polymerase which appears to share many similarities
to pause in its elongation [65]. Rho-depen- with the bacteriophage lambda system ([69]
dent sites (rut sites) are cytosine-rich and and references therein).
guanosine-poor [260] but they do not have
the degree of sequence conservation asso-
ciated with rho-independent sites. Although 9.3 EUKARYOTES HAVE THREE
many can be drawn as stem-loop structures, DIFFERENT NUCLEAR RNA
the base-pairing tends to be relatively POLYMERASES
unstable and largely involves A: U base-
pairing. Indeed, a considerable body of Transcription in eukaryotic nuclei is per-
evidence has accumulated showing that rho- formed by three separate enzymes [70-74].
dependent termination is enhanced by RNA polymerase I is located in the nucleoli,
decreased secondary structure in the RNA transcribes the genes for rRNA and is
transcript [65] and that rho binds to nascent responsible for 50-70% of total RNA
mRNA that is relatively free of secondary transcription. RNA polymerase II occurs
structure [66]. Models for rho-dependent in the nucleoplasm and synthesizes mRNA
termination [46, 50, 67, 68] propose that precursors and most of the U-series of small
the factor first binds to nascent RNA that nuclear RNAs (snRNAs). It accounts for
lacks secondary structure. It may then move 20-40% of total RNA transcription. RNA
along the transcript until it finds a paused polymerase III is also located in the nu-
RNA polymerase and this movement cleoplasm and transcribes a series of small
could require the hydrolysis of nucleoside RNAs. These include RNA species required
triphosphate [46]. Alternatively, the hydro- for protein synthesis (tRNA and 5S RNA),
lysis of NTP may result from a conform- one of the snRNAs required for mRNA
ational change in rho brought about by processing (U6 snRNA), a small RNA
the interaction of the nucleotide with the required for protein transport (7SL RNA),
ternary complex. This in turn could cause RNAs required for the regulation of viral
the release of the nascent RNA chain ([62] gene expression (adenovirus VA RNA and
and references therein). Epstein Barr Virus EBER RNA) and other
It seems likely that there are a number small RNA species of unknown function,
of factors that bring about termination in such as 7SK RNA. RNA polymerase III
prokaryotes. Nus A protein, which is con- synthesizes approximately 10% of total
sidered in chapter 10 under the control transcribed RNA.
of termination (section 10.2.2), is a ter- All three enzymes catalyse RNA synthesis
mination factor that, in bacteriophage on a DNA template as previously described
lambda, interacts with an anti-termination for the E. coli enzyme and they require a
factor N. Anti termination is a process divalent metal ion which can be Mg 2 + or
whereby RNA polymerase is rendered Mn 2 +. When assayed in vitro, polymerase
insensitive to some termination mechanisms. II is much more active with Mn 2+ and
The best-known systems in which it has polymerase III is slightly so. However,
been studied are; the regulation of gene concentrations of Mn2+ in vivo are low
expression in bacteriophage lambda (section and there is evidence that the use of MnZ+
10.2.2); the attenuation of amino acid in vitro may alter the binding properties of
biosynthetic operons (section 10.2.1) and the enzyme [75]. In the analysis of enzyme
Nuclear RNA polymerases 351
activity, use is also made of their differing genuine subunit that is differentially lost
sensitivity to the fungal amatoxins (a-, P- during enzyme purification. If a subunit
and y-amanitin, amanin and amanullin) functioned as a factor, this could also
of which the most commonly used is a- explain low abundance. A factor like sigma
amanitin. RNA polymerase II is inhibited in the E. coli enzyme, that is only required
by a-amanitin at SOng/mi. RNA polymerase at certain points in the transcription cycle,
III is much less sensitive but is inhibited at would not necessarily occur in stoichiometric
concentrations of 5 pg/ml whereas RNA amounts on the purified enzyme.
polymerase II is insensitive. De Mercoyrol Since it is not yet possible to reconstitute
[76] has demonstrated that a-amanitin does active enzyme from its subunits, the func-
not block the formation of the first phos- tional significance of any component is hard
phodiester bond but inhibits the catalytic to ascertain but considerable progress is
accumulation of trinucleotide, perhaps being made with the recently developed
because it blocks an isomerization step method of epitope tagging. In the first stage
required for translocation after phospho- of this technique, a gene encoding a poly-
diester bond formation. As with prokaryotic merase subunit is modified so that, during
RNA polymerase, all three eukaryotic protein synthesis, an extra string of amino
enzymes contain bound zinc, probably acids is inserted on the end of the poly-
associated with a conserved amino acid peptide. The extra amino acids are chosen
sequence in the largest subunits [77]. so as to minimize disruption of the protein
Sedimentation analyses show that all and, most importantly, so that they are
three enzymes are very large with Mr values recognized by a well-characterized mono-
of 500000-600000 and they contain up to clonal antibody. The enzyme can then be
15 subunits. Tabulation of the components purified by gentle immunoaffinity methods
of the three polymerases from various and the subunit composition compared
sources [70] reveals substantial interspecies with that derived after more traditional
variation in the Mr and numbers of the purification techniques.
subunits. Thus polymerase III has been Perhaps the most studied enzyme is
reported to have from 9 to 15 subunits RNA polymerase II which has been puri-
depending on the species from which it is fied to near homogeneity from more than
isolated. In general terms, however, each 20 different species. That of the yeast,
enzyme possesses two non-identical, large Saccharomyces cerevisiae, has ten subunits
subunits of Mr 120000-220000 and up to (RPBl-RPBlO) whether purified by con-
13 smaller subunits which, with the ex- ventional techniques or by immunopre-
ception of an 80000-90000 subunit in cipitation with a monoclonal antibody
polymerase III, have Mr of less than 50000. against epitope-tagged subunit 3 [79]. Each
Lewis and Burgess [78] have discussed the subunit is encoded by a single copy gene but
difficulty of ascertaining the precise number the enzyme appears to be composed of one
of subunits in the active enzymes. Some polypeptide of each of RPBl, 2, 6, 8 and 10,
subunits may only be present in the purified two copies of each of RPB3, 5 and 9, and
enzyme at a molar ratio of less than one less than stoichiometric amounts of subunits
molecule per enzyme molecule. Several RPB4 and 7.
explanations are possible for such a lack of The two largest subunits of the enzyme
stoichiometry. The polypeptide concerned contain extensive regions of homology with
could be a contaminant or it could be a the p and P' -subunits of E. coli RNA poly-
352 RNA biosynthesis

merase [80] and are related in size and in RNA polymerases I, II and III [86]. It
sequence to the large subunits of RNA would thus appear that many of the enzymic
polymerases I and III [73]. The largest components are substantially preserved
subunit of 220 kDa is the largest of any of between the three eukaryotic enzymes and
the RNA polymerases and experiments indeed exhibit considerable similarities with
involving cross-linking, together with prokaryotic RNA polymerase.
susceptibility to antibodies and proteases,
indicate that it is involved in binding to the
DNA template [71]. It contains, in addition 9.4 THE INITIATION OF EUKARYOTIC
to the regions of homology with other TRANSCRIPTION
enzymes, a carboxy-terminal domain (CTD)
that forms an extension or tail to the core The study of eukaryotic RNA polymerase
enzyme and consists of tandem repeats of a activity has been much enhanced by the
seven amino acid sequence: Tyr-Ser-Pro- development of systems in which the en-
Thr-Ser-Pro-Ser (reviewed in [81 ]). The zymes correctly transcribe specific genes
tandem repeats are common to all known in vitro. The first of these was a transcription
RNA polymerase II enzymes but the system for polymerase III [87] and led to
number of repeats varies from 17 in the the use of extracts of Xenopus oocytes as a
malarial parasite to 52 in mammals. The major system to support transcription by
extension is essential for enzyme function this enzyme [88, 89]. Similar systems,
and may be unphosphorylated (RNA employing extracts of tissue culture cells,
polymerase IIA) or extensively phosphory- were also described for the transcription of
lated (RNA polymerase 110). Phosphory- cloned eukaryotic genes and of viral genes
lation causes .a conformational change in by both polymerases II and III [90, 91 ]. The
the carboxyl domain [261] and recent data last to be developed was a cell-free extract
indicate that polymerase IIA forms the that correctly initiated the transcription
initiation complex but that phosphorylation of pre-rRNA from cloned rDNA [92].
of the C-terminal domain occurs before the Such polymerase !-dependent systems are
initiation of transcription [82]. There is also species-specific [93].
evidence that transcription factors could Transcription is also studied by the
interact with the domain [83]. microinjection of cloned eukaryotic genes
The next largest subunit of RNA poly- into Xenopus oocytes [94], by the stable
merase II, RPB2, appears to participate in introduction of genes into mammalian cells
substrate binding [85] and phosphodiester in culture [95] and by the insertion of cellular
bond formation, thus showing functional genes into an SV40 viral vector [96] or a
homology with the P subunit of bacterial plasmid containing the SV40 or other viral
enzymes. The third largest subunit is also replication origins [97].
an essential component of the transcription The use of such systems has shown that
apparatus [84], may be the equivalent of RNA synthesis by eukaryotic polymerases
subunit RPCS that is common to RNA has much in common with that catalysed
polymerases I and III, and is probably the by the prokaryotic enzyme. Since there are
functional homologue of prokaryotic sigma three nuclear enzymes, however, one may
factor [84]. At least three other subunits, expect differences between the three in
RPBS, RPB6 and RPB8, are immuno- promoter recognition as well as termination
logically and biochemically indistinguishable and control of transcription.
Initiation of eukaryotic transcription 353

Promoror

Enhancer Upsrream TATA ....,__srrucrural __ _

elemenr elemenr box gene

-~~~H ~~ ~~-
~ -110 to-40 t -30
variable disrances
t
Transcriprion
srarr sire

Fig. 9.6 A diagram of the promoter region for RNA polymerase II.

9.4.1 Initiation by RNA polymerase II helical turn) from the start site of pro-
karyotic transcription. Second, there is
The promoter for RNA polymerase II a degree of sequence conservation in
consists of a number of sequence elements the prokaryotic start site but much greater
required for accurate and efficient initiation variability in eukaryotes. Transcriptional
of transcription (reviewed in [71 ]). Two of analysis and S1 nuclease mapping have
these show similarities with the conserved shown that a functional TAT A box precedes
sequences of prokaryotes. The first con- most genes transcribed by RNA polymerase
served element, identified when the 5'- II. There are, however, genes, such as the
regions of eukaryotic gene sequences late SV40 genes and U1 snRNA genes, that
were compared, was originally named the contain equivalent sequences that can at
Goldberg/Hogness box after its discoverers best only be described as TATA-like [99,
[98]. It is now usually called the TATA box 100]. They may form another class of
and is an AT-rich region with the consensus promoter. Other genes that lack TAT A
sequence: boxes are the so-called housekeeping genes
that encode constitutively expressed pro-
5' TATA*A* 3' teins that are continuously expressed but at
It occurs approximately 30 bp upstream a low level [101]. The genes have a CG-rich
from the transcriptional start site in most promoter that is discussed in section 10.6.2
eukaryotes (Fig. 9 .6) but 60-120 nucleotides and their transcripts have multiple 5'-ends.
upstream of the start site of yeast genes. It The second element in the RNA poly-
has been called the selector sequence and its merase II promoter is the so-called upstream
function is to fix the location of the start promoter element (UPE). It occurs in most
site. Mutations in the consensus sequence protein-encoding genes and consists of one
can profoundly affect correct initiation. or more 8-12 nucleotide segments located a
Conversely, nucleotide deletions to the variable distance ( -40 to -110) upstream
transcription start site region result in of the transcription start site (Fig. 9.6).
initiation at a new site, still approximately Whereas the function of the TAT A box is to
30 bp downstream from the TAT A box. ensure accurate initiation of transcription,
Although this element is obviously very the UPE increases the rate of transcription.
similar to the prokaryotic Pribnow box two One common variant of the UPE is the
differences are thus immediately apparent. so-called CAAT or CCAAT box which has
First, the Pribnow box is 10bp (one DNA the consensus sequence:
354 RNA biosynthesis

5' GGtCAAlCT 3' containing a promoter sequence. They are

then often identified by DNA footprinting
and occurs 70-90 bp upstream from the and gel retardation assays (section A.9.2)
start site. A further variant is a GC-rich and, in an increasing number of cases, they
sequence (GC box) with the consensus: have been substantially purified by recog-
nition-site affinity chromatography. In some
5' CCGCCC 3'
cases, their genes have been cloned.
or its complement: The use of soluble, cell-free systems to
transcribe a DNA template with a minimal
5' GGGCGG 3'
TATA box promoter led to the identi-
which can occur in one or more copies fication of sets of transcription initiation
[102, 103] and may be present in addition factors from various species and cell types.
to a CAAT box as the so-called -100 Inevitably, this resulted in multiple names
element [104]. and acronyms that without doubt describe
More complex regulation of transcription the same or closely related proteins. Table
is provided by enhancer elements (Fig. 9.6). 9.1 presents the most generally accepted
These are short stretches of nucleotides names that will be used for the remainder of
which act in cis to increase the transcription this discussion and also lists the probable
of nearby genes. They function relatively functional equivalents that have been
independently of position, orientation and described from other systems.
distance. Some show narrow tissue and The pivotal factor in the recognition of
temporal specificity whereas others permit the T ATA box is most commonly called
expression in many cell types. TFIID (transcription factor D for RNA
Although presented above as three polymerase II). It binds the TATA box
separate elements, the distinction between independently, without the involvement of
promoters, upstream elements and en- other factors or RNA polymerase ([111]
hancers is blurred. They share many prop- and references therein). It is also apparently
erties and often overlap, with the same the factor that recognizes and binds to
consensus sequence serving as both pro- promoters with little TATA box homology
moter and enhancer. It will be convenient [112]. TFIID has been described as a com-
here, however, to discuss the role of TATA mitment factor as its binding is a prerequisite
boxes and UPEs in the initiation of tran- for the formation of the basal transcription
scription while leaving enhancers and their apparatus (Fig. 9.7) that includes RNA
role in the regulation of transcription to polymerase II and several more factors:
be considered under the control of gene TFIIA, TFIIB, TFIIE (reviewed in [113]).
expression (chapter 10). Of these, TFIIA stimulates and facilitates
Most, probably all, of the above sequence the binding of TFIID but is not always
elements are targets for proteins. Indeed, required. TFIIE is multicomponent and has
eukaryotic RNA polymerases are unable to been separated into TFIIE and TFIIF of
recognize promoters without the aid of which the latter consists of two polypeptides
proteins. Often called transcription factors, of 30 kDa and 78 kDa. Impure TFIIF has
these proteins are frequently present at very helicase activity [109] but this is not present
low concentrations but they are initially in pure FC which is probably the same
detected in cellular fractions required for protein [108]. More recently, a further
transcription in vitro from a DNA template factor, TFIIG, that is also an essential
Initiation of eukaryotic transcription 355

Table 9.1 Transcription initiation factors for RNA polymerase II

Most Other names or probable functional

common equivalents in various eukaryotic
name systems Function Refs
-------
Band D Formation of basal
TFIID TATA factor, BTFl, DB } FD and FF transcription complex
Stimulates TFIID binding but ) [71, 105,107]
TFIIA STF, AB r and e
not always required
TFIIB BTF3, CBI, a } FB +FE Stabilization of
TFIIE Multicomponent and including initiation complex.
: TFIIE + [ TFIIF (30 k + 78 k) ] May include DNA (71, 106,
RAP 30/74 helicase activity (109]. 107108,
FC30+80 The boxed components 110]
BTF2 are probably the same
fly two polypeptides.

component of the transcription complex, to do so by interaction of the complex with

has been identified [262]. TFIID [117). There are multiple CAAT-
TFIID is absolutely required for tran- binding proteins each of which binds to
scriptional initiation and its binding is stable partially overlapping sets of genes [263)
through several rounds of transcription but whether they all interact with TFIID is
[114). Both binding [111) and the activation unknown. Other, more specific transcrip-
of transcription [115) are dependent on a tion factors, are, however, thought to act
large C-terminal region of the protein that through TFIID. These include the herpes
stretches from residue 63 to 240. Within this virus activator protein VP16 [118), GAL
is a highly basic domain (residues 120-156) 4 that regulates the genes encoding the
and a region (residues 197-240) that has galactose-metabolizing enzymes of yeast
sequence similarity with the -to-binding [119] and the mammalian activator protein
region of prokaryotic sigma factor [111, ATF [264).
116).
As well as being pivotal in the formation
of the basal transcription factor, TFIID also 9.4.2 Initiation by RNA polymerase Ill
plays a role in the modulation of transcrip-
tion. A transcription factor SP1 interacts (a) Internal promoters for RNA polymerase
with the GC box of the UPE and in so doing III
causes a fivefold increase in transcription.
Evidence collected by Pugh and Tjian [117) The promoters for the tRNA and 5S rRNA
suggests that the complex of the GC box and genes do not lie in their 5' -flanking se-
SP1 causes stimulated transcription by quences but within the coding region of the
interacting with TFIID promoter complex genes themselves (reviewed in [73, 120,
via a co-activator (Fig. 9. 7). Similarly, the 121 ]). This unexpected finding resulted from
factor CTF activates transcription when it the work of Brown and colleagues on the
binds to the CAAT box and is again believed expression of Xenopus 5S RNA genes in
356 RNA biosynthesis

----1 GC Box ~1-------i[TATA Box~[---!__ _S_t_ru_c_tu_ra_lg~e_n_e

j Formation of
committed complex

O®
JFifoi:D
----1 GC Box [ [ ---t.__ _S;;. ;t;. ; ru;. ; .ct.;.; u. .;ra;.;. .g:ii:.,;e;.;..n~e
[TATA Box .... l

Formation of basal
transcription complex

Formation of activated
transcription complex

ene

Fig. 9.7 Diagrammatic representation of initiation by RNA polymerase II.

oocytes and in systems for transcription scription. Furthermore, when they deleted
in vitro. They found that the entire 5'- the 5'-end of the coding sequence, they
ftanking sequence of the gene could be found that 5S-sized RNA was still made
removed without the loss of 5S RNA tran- from a new start site that corresponded to
Initiation of eukaryotic transcription 357

cfWA1
58 rRNAgene

----1~U~r{s~ A - B ~------
tRNAgene

------I~ A-B -~-

VAigene

~---
U6 snRNA gene

--{§------ ~'.LLC..L..'-LLL£.£.£..£LLLLUI-~ ~
7SKRNAgene
Fig. 9.8 The promoter structure for genes transcribed by RNA polymerase III. The boxes A, B, and C
are the internal promoter elements of 5S RNA, tRNA and VAI genes. The dotted outline boxes, URS
represent upstream regulatory elements that are present in the flanking sequences of some 5S and
tRNA genes. TATA indicates the presence of RNA polymerase 11-like TATA boxes. PSE and DSE are
proximal and distal sequence elements of the U6 and 7SK genes.

where the 5'-end of the gene had been. Not cognized by RNA polymerase III [124] but
until 50 coding nucleotides had been deleted by a transcription factor, known as TFIIIA
was there a drop in the efficiency of tran- [125], which specifically interacts with two
scription [122]. Subsequent experiments, in factor-binding domains at the boundaries of
which the 3'-end of the gene was deleted the promoter region [126]. These domains
[123], or in which extra nucleotide sequences have become known as Block A and Block
were inserted into the gene [122], showed C in reference to the two-block promoter of
that the synthesis of 5S RNA was controlled tRNA genes (see below).
by a promoter located between residues 50 TFIIIA was the first eukaryotic tran-
and 83 of the Xenopus gene. The promoter scription factor to be discovered and is the
is known as the internal control region or archetype of a group of proteins that interact
JCR (Fig. 9.8) and is required and sufficient with DNA through so-called zinc fingers
for transcription. It is not, however, re- (section 10.5.2). The protein has nine of
358 RNA biosynthesis

Fig. 9.9 The relationship of the split promoter of tRNA genes to the clover-leaf tRNA secondary
structure. The shaded portions of the tRNA molecule and the labelled invariant nucleotides are
transcribed from the internal promoter.

these Zn2+ -binding domains that jointly shown that they too have an internal pro-
interact with approximately five helical moter (Fig. 9.8) but in this case it is clearly
turns of the ICR. The interaction is strongest split into two blocks termed A and B [129].
with box C (base pairs 80-97) but also Block A extends from nucleotides 8 to 19
occurs with box A (bp 49-60) and weakly and includes the portion of the gene that
with some intervening regions. Most of the encodes the tRNA D loop and four invariant
zinc fingers and the DNA binding activity nucleotides. The B block runs from nucleo-
are in the N-terminal portion of the poly- tides 52 to 62 including those encoding the
peptide but this portion of the protein T-loop and five invariant nucleotides (Fig.
cannot by itself bring about formation of a 9.9). Thus, the invariant nature of some
stable transcription complex. Neither will it tRNA nucleotides appears to be important
permit the cooperative binding that appears both in tRNA tertiary structure (section
to occur between the tandemly arranged 5S 12.3.1) and in the initiation of tRNA syn-
RNA genes [127]. The binding of TFIIIA thesis. Because of the length heterogeneity
is the first stage in the formation of a 5S of the tRNA variable loop and the presence
transcription complex and is followed by the in some tRNA genes of inserts, the distance
sequential binding of TFIIIC and TFIIIB between the A and B blocks in the gene can
[128]. It is the complete complex that is vary between 25 and 95 bp.
recognized by RNA polymerase III [128]. Block B has no equivalent in the 5S gene
Similar studies with tRNA genes have intergenic promoter but block A is func-
Initiation of eukaryotic transcription 359

tionally equivalent to the 5S block A. (b) Upstream promoters for RNA

Conversely, there is no equivalent to the polymerase Ill
C block in the promoter of tRNA genes
which are not therefore targets for TFIIIA. Although the internal promoters of 5S
Instead, the first step in the assembly of and tRNA genes were unexpected, they
a transcription complex on tRNA genes nevertheless form a consistent picture
occurs with the binding of TFIIIC to the whereby the sequential recognition of the
B block of the promoter. The factor also promoter by transcription factors leads to
interacts with the A block. Proteolysis of the the accurate positioning of RNA polymerase
yeast TFIIIC (also called Tau) shows that III. The adenovirus VA RNA gene, which is
the 300 kDa protein has two functional transcribed by RNA polymerase III into a
domains one of which binds to B block RNA that regulates viral gene expression,
and the other to A block [130]. Electron supports the concept. Like tRNA genes,
microscopy indicates that between the two it has A and B block internal promoters
domains the protein is very flexible allowing (Fig. 9.8). However, it has recently become
it to adopt a globular or dumbbell shape clear that this picture of RNA polymerase
depending on the separation of the A and III transcription is too simplistic. Many 5S
B blocks between the 25 and 95 bp minimum genes have been found to have 5'-sequences
and maximum spacing[131]. There is evi- that dramatically influence the rate of their
dence, in at least some systems, that TFIIIC transcription. Furthermore, the elements
is multicomponent and may include a poly- involved are homologous in sequence and
peptide that specifically recognizes tRNA location with the T A TA box of the RNA
promoters [132]. However, the data are polymerase II promoter [135]. Other genes
contradictory and await clarification (re- that are transcribed by RNA polymerase III
viewed in [121]). As with the 5S promoter, blur the picture even more dramatically
the binding of TFIIIC is stabilized by TFIIIB in that they have no internal promoters
so forming a pre-initiation complex that is (reviewed in [136]). Thus, U6 snRNA and
recognized by RNA polymerase III. 7SK RNA genes have TATA boxes together
TFIIIB is the protein that correctly with other upstream elements that are more
positions RNA polymerase III at the start usually associated with the enhancement of
site for the initiation of transcription of both RNA polymerase II transcription (Fig. 9.8).
5S and tRNA genes [133]. Kassavetis et a/. These include proximal elements, that may
demonstrated that, once TFIIIB is asso- function to position TFIIIB and thus RNA
ciated with the complexes, the factors polymerase III [137], but are nevertheless
TFIIIA and TFIIIC can be removed with homologous to the proximal elements of the
heparin or high salt, leaving TFIIIB still snRNA genes transcribed by RNA poly-
attached. The partially stripped promoters merase II. In both Xenopus and plants
still permit the accurate initiation of tran- it appears that the distance between the
scription by RNA polymerase III [133]. TAT A box and the upstream element in
Thus, TFIIIA and TFIIIC function only as snRNA genes is critical in determining
promoter recognition factors that sequester whether they are recognized by RNA
TFIIIB. Footprinting analyses, that show polymerase II or III [138, 270]. The pro-
that TFIIIB protects 5' -flanking sequences moters of U6 and 7SK genes also include
of genes rather than the internal promoters, distal elements that may be equivalent to
support these concepts [134]. enhancers (Fig. 9.8). In 7SK genes these
360 RNA biosynthesis

include an essential CACCC motif that is synthesis and is regulated in response

similar to several eukaryotic regulatory to changes in growth and metabolism
elements [139]. Similarly, the distal element (reviewed in [74, 265]). The high tran-
of U6 snRNA genes includes an octamer scription rate may be presumed to reflect
motif like that controlling the transcription many features of the rONA genes. There
of immunoglobulin and histone genes by is a high gene dosage and, from yeasts to
RNA polymerase II [140]. Clearly these man, the rONA is arranged in multiple
findings cloud the differences between the tandem copies with the coding regions
two polymerases and suggest that they separated by intergenic spacers (section
evolved from a common ancestor. 8.4.3). The genes are concentrated in the
nucleolus as is RNA polymerase I, the
(c) TFIIIA may regulate the expression of5S enzyme that apparently transcribes only
rRNA genes pre-rRNA. Transcriptional regulation is
focused in the intergenic spacer (formerly,
There is evidence that the developmental but mistakenly, called the non-transcribed
regulation of oocyte and somatic 5S RNA spacer). It contains at least three controlling
gene expression in Xenopus may be medi- elements; proximal promoters, upstream
ated through transcription factor TFIIIA. promoter elements and terminators. In
Oocytes express 5S RNA from the abundant many cases, upstream sequences also in-
oocyte 5S genes in the presence of high clude elements with many of the properties
concentrations of TFIIIA whereas somatic of the enhancers of RNA polymerase II.
cells have low concentrations of TFIIIA and RNA polymerase I was the last of the
transcribe 5S RNA from the low abundance eukaryotic nuclear RNA polymerases for
family of somatic 5S RNA genes. Wolffe which in vitro and in vivo transcription
and Brown [141] have explained the dif- systems were developed [143], in part
ferential regulation by differences in the because, unlike polymerase II and III, this
stability of the TFIIIA-ONA complexes. enzyme is very species-specific. Thus,
They suggest that, because the somatic mouse RNA polymerase recognizes its own
promoter-TFIIIA complexes are more promoter complex and that of closely
stable, they are able to compete more related species like rat but it will not re-
effectively for the declining quantities of the cognize that of human. Similarly, the human
factor that are present after the fertilization enzyme will recognize human and rhesus
of the oocyte. However, Blanco et al. [142] transcription systems but not that of mouse
have reported two different TFIIIA ac- [144, 145]. Presumably, the singular speci-
tivities in this system. They claim that ficity of the enzyme has permitted this
transcription of oocyte 5S RNA requires evolutionary drift.
a 39 kOa form of the factor whereas a Notwithstanding species specificity,
42 kOa form activates the somatic genes rONA promoters of vertebrates share many
and represses the oocyte genes. common features (Fig. 9.10). In mammals,
the core promoter element (CPE, also called
the proximal promoter) overlaps the tran-
9.4.3 Initiation by RNA polymerase I scribed gene and spans from -31 to +6
with respect to the transcription start site.
The transcription of pre-rRNA represents A second distinct but interacting domain,
approximately half of all cellular RNA called the upstream control element (UCE)
Initiation of eukaryotic transcription 361

(a) The lntergenic Spacer of Xenopus laevis rONA

Spacer Spacer
Terminators promoter promoter Gene
(BAM Island) (BAM Island) Terminator promoter
t2
~ t3 ~ ~
~ ~
.._____,____., ~
60/81 Repeats 60/81 Repeats
(enhancers) (enhancers)

1-----------2300-5300bp----------~

(b) The lntergenic Spacer of mouse rONA Spacer Gene

Terminators promoter Terminator promoter
WH ~ ~ +
28~ -25kb.~/-----ID------·---.-- ~
140 bp Repeats
(enhancers)

1 - - - - - - - - - - - - - -30000bp -----------~
Fig. 9.10 The intergenic spacers ofrDNA from (a) Xenopus laevis and (b) mouse. The black bar in the
Xenopus spacer promoters indicates that enhancer elements occur within them. ETS is the external
transcribed spacer (section 8.4.3).

or upstream promoter element (UPE) is transcripts terminate upstream of the

located between -107 and -187 [146, 147]. true gene promoters (reviewed in [265]);
The CPE is necessary for transcription and however the function of the transcripts, if
in vitro is sufficient for basal rates of rRNA any, is a matter of speculation.
synthesis. The UPE is required, however, In addition to the promoter-like elements,
for efficient transcription both in vitro and the Xenopus intergenic spacer contains
in vivo [148-150]. The intergenic spacers of repetitive short sequences, the 60/81 bp
some species contain a series of repetitive elements, that are clustered in tandem
sequences upstream of the promoter that (Fig. 9 .10) and which stimulate transcription
are, in effect, multiple duplications of when placed at a variable distance from the
promoter elements. Thus, in Xenopus, the promoter and when placed in either orien-
core promoter again spans the transcription tation [153, 154]. They are thus analogous
start site ( -14 to +4) but in addition there in their activity, but perhaps not in their
are multiple promoter-like elements that are mechanism, to the enhancers of RNA
approximately 90% homologous to the polymerase II (section 10.4). A 140bp
promoter and are located in the so-called repeated element between the core and
Bam islands (Fig. 9 .10). Drosophila likewise upstream promoters of mouse rDNA
has multiple internally repetitious promoters (Fig. 9 .10) has also recently been shown to
in its intergenic spacer [151, 152]. Tran- have an enhancer-like function [155] and
scription can initiate from within these enhancing elements have also been detected
intergenic promoters and the resultant in rat and Drosophila [265].
362 RNA biosynthesis

At least two protein transcription factors appears that species specificity resides in
are required for pre-rRNA synthesis. In rat, SLl and its interaction with both UBF and
footprinting experiments have shown that DNA. The Xenopus 60/81 enhancer repeats
the factors SLl and SF1 interact with both (Fig. 9.10) exhibit considerable sequence
the core and upstream promoters. Binding homology with the UBF-binding region
to and transcription from the proximal [266] and apparently act by enhancing
promoter is much more efficient than that the formation of UBF-SLl transcription
from the upstream elements [156] so it is complexes [267].
uncertain what the function of the upstream In addition to detecting factors that may
promoters may be. Moss [157] has suggested be homologous to those described above,
that they function as sinks for RNA poly- three groups have detected factors that
merase but it is also possible that they are interact with RNA polymerase I [166-168].
alternative promoters the use of which is That called TIF-1A by Grummt and col-
associated with differential control of rDNA leagues, interacts with the enzyme and
gene expression. converts it into an active holoenzyme that
Rat SF1 is apparently homologous with has rDNA promoter specificity [167]. It thus
the factor called UBF in human cells and behaves like bacterial sigma factor. These
with xUBF of Xenopus [158, 159]. The workers have also shown that dephos-
proteins recognize the same DNA sequence phorylation of RNA polymerase I abolishes
elements [160] and cannot therefore be its specificity for rDNA promoters. Another
responsible for species specificity of pro- factor, designated TFIC by Thompson and
moter recognition. Factor SLl, on the other coworkers [168, 169] is also tightly asso-
hand, is a good candidate for the source of ciated with RNA polymerase I and reduced
species specificity. Human SLl (hSLl) will levels of it have been implicated in the
cause a mouse in vitro transcription system reduced transcription of rRNA in gluco-
to recognize a human promoter [161]. corticoid-treated lymphosarcoma cells.
Similarly, rat SLl ( rSLl) will reprogramme A further controlling influence on the
a human transcription system to recognize a initiation of transcription from tandemly
rat promoter [156]. Bell et al. [162] have arranged rRNA genes are the terminator
shown that mouse and human RNA poly- sites of the previous gene which, in addition
merase I and UBF are functionally inter- to their termination activity, are in some
changeable but SLl are distinct in both their cases able to stimulate the adjacent gene
binding and transcriptional activities. Thus, promoter (reviewed in [170]). Thus, it is
although mouse SLl will selectively interact now known that, in Xenopus, RNA poly-
with DNA in the absence of UBF [162], merase I transcribes right across the inter-
human SLl does not recognize DNA and genic spacer to a terminator called t3 that
associates with the promoter only by inter- is approximately 60 bp upstream of the
action with UBF [163]. A strong cooperative promoter for the next transcription unit
complex between the two proteins is critical (section 9.5.3). It has been suggested that
to transcriptional activation [162, 164]. UBF it may act with the promoter in an inter-
has been purified from both human and dependent complex bringing about the
Xenopus cells and shown to have multiple termination of one transcription unit and
domains. The DNA binding domains exhibit the initiation of the next. Mammalian RNA
the same specificity but the domains that polymerase terminates after transcribing a
interact with SLl differ [165]. Thus, it much shorter distance into the intergenic
Termination of transcription in eukaryotes 363

spacer (section 9.5.3) but there are again (processing is considered in sections 11.5,
extra terminator sequences close to the next 11.6 and 11.7).
promoter. They may well have two func-
tions; the termination of transcripts that
have initiated within the intergenic spacer 9.5.1 Termination by RNA polymerase II
and exerting a positive influence on the
initiation of the next transcription unit. It is (a) Polyadenylated mRNA
suggested that RNA polymerase is 'handed
over' from the terminator to the promoter. Use of the run-off transcription assay
However, recent experiments by Lab hart (section A.ll) has demonstrated that
and Reeder (171] have demonstrated that RNA polymerase II characteristically
preventing the enzyme from reaching the terminates at heterogeneous sites up to
Xenopus t3 terminator had no effect on several thousand base pairs beyond the
the density of polymerase molecules that polyadenylation site (172]. Thus, the ter-
collected at the 5'-ends of genes. mination of mouse P-globin occurs about
1 kb past the end of the gene (173] whereas
mouse a-amylase transcripts terminate at
9.5 TERMINATION OF multiple sites 2-4 kb past the 3'-terminus
TRANSCRIPTION IN EUKARYOTES (174]. Proudfoot (175] has tabulated the
run-off characterized termination of other
Termination of transcription in eukaryotes genes and has reviewed termination by
is not well understood. Often it is difficult to RNA polymerase II.
even be sure of the relationship between Section 11.5 .1 describes how two se-
observed 3'-termini of newly made RNA quence elements of polymerase II tran-
molecules and the real point at which scripts, the AAUAAA and a GC-rich
transcription terminates. This is especially sequence motifs, are specific recognition
so with the transcripts of RNA polymerase signals for the 3'-processing of pre-mRNA.
II, the majority of which are post-transcrip- It has become clear that these same elements
tionally modified by 3'-polyadenylation. are also involved in transcriptional ter-
The only method currently available to mination. This was first indicated in a
identify termination sites is run-on or run-off patient with a-thalassaemia in whom a base
transcription (both names are confusingly change in the a2-globin gene converted the
used for the same process). By using purified AATAAA motif into AATAAG. This not
nuclei for transcription in vitro, the synthesis only inactivated the processing of a2-globin
of partially, transcribed and still nascent pre-mRNA but also caused transcription to
RNA molecules is continued with the continue well past the normal termination
incorporation of radioactive nucleotides. sites (176]. Similarly, the AATAAA el-
After purification, the radioactive RNA is ement of a mouse globin gene could be
hybridized to fragments of DNA that used in a gene construct to promote the
correspond to the 3'-end and the 3'-ftanking termination of the adenovirus E1A gene
sequences of the mRNA encoding sequence. but failed to do so if changed by point
In this way, it has been demonstrated that mutation (177]. Hybrid gene constructs in
many genes are transcribed well past their SV40 and polyoma have also been used to
apparent 3'-termini and are then subject to demonstrate that termination requires a
processing to generate their 3'-termini functional 3'-processing site and that both
364 RNA biosynthesis

the AAT AAA and the GC-rich elements is a preserved motif GmN 0_3AAA
were required [178, 179]. Furthermore, 0 / ANNAGA in the immediate 3' -flanking
the stronger the processing site the more region of snRNA genes. The second is an
efficient was the termination. internal sequence close to the 3' -end of all
Two models have been put forward snRNA genes that is potentially able to form
to account for the coupling between 3'- a stem-loop structure and the third element
processing and termination. Darnell and is the promoter of snRNA genes. The role of
coworkers suggest that a specific elongation the promoter elements is not understood but
factor could be released from RNA poly- snRNA termination signals do not work if
merase II during its passage over the pro- combined in gene constructs with non-
cessing signal sequences. This, they suggest snRNA promoters. For example, RNA
would destabilize elongation and cause polymerase II initiating at an adenovirus
random termination [177]. The model most late promoter, will transcribe through an
supported by the available evidence, snRNA termination sequence to terminate
however, is that the 3' -terminus is formed by at a downstream polyadenylation site [182].
the endonucleolytic cleavage of a transcript It thus seems likely that initiation at a
while it is still being synthesized. Cleavage is snRNA promoter is accompanied by the
anticipated to occur at the processing site incorporation into the initiation complex of
while RNA polymerase is still advancing a a specific factor that then promotes dis-
considerable distance downstream [172, engagement of polymerase at the snRNA
178]. After cleavage, the newly formed termination site.
3' -terminus becomes the site for poly-
adenylation (section 11.5.1) while the new (c) Histone genes
5' -terminus is not capped and is vulnerable
to degradation by exonuclease. Proudfoot Histone genes illustrate a third type of
[175] has suggested that three factors may termination by RNA polymerase II. As
contribute to the eventual termination of described in section 11.5.2, the 3'-processing
transcription and release of the polymerase. of histone transcripts involves the recog-
These are the exonuclease activity, a RNA: nition of a series of conserved elements by a
DNA helicase that might be required to number of factors that include snRNA U7.
unwind the nascent RNA:DNA hybrids and Termination of transcription occurs well
pausing sites on the DNA required to slow downstream of these processing sites and,
the progress of RNA polymerase. in contrast to polyadenylated pre-mRNA,
the two processes can be independent of
each other. Thus, the conserved processing
(b) snRNA genes
signals of some histone genes can be re-
moved without affecting termination and it
Termination of the transcription of the has recently been shown that termination
genes for snRNAs (snRNAs themselves occurs in an A-rich region [183]. The motifs
are described in section 11.2) appears to are close to but separate from the processing
occur at or close to the 3'-terminus of the signals and, when placed in a globin gene
mature snRNA [180, 181 ]. Three sequence intron, they cause premature termination
elements are apparently required for this of transcription. In contrast to these data
precise recognition event. The first of these however, recent studies with the mouse
Termination of transcription in eukaryotes 365

H2A gene have shown that a processing site bond energy to melt RNA:DNA hybrids. It
is required for termination [268]. thus resembles the prokaryotic termination
factor, rho (section 9.2.1).

9.5.2 Termination by RNA polymerase Ill

9.5.3 Termination by RNA polymerase I
Termination of 5S RNA transcripts by RNA
polymerase III occurs in a single consensus The ribosomal genes of Xenopus have three
sequence that consists of a run of four or major sites that have been associated with
more T residues in the non-coding strand RNA 3'-end formation and are designated
and is surrounded by GC-rich sequences tb t 2 and t 3 (Fig. 9.10). Their relationship
[184]. Similar terminator sequences occur with termination was first demonstrated by
immediately or soon after the coding regions Labhart and Reeder [192] who showed that
of almost all known tRNA genes, and t 1 was at the 3'-end of 28S RNA but was
mutations that create strings of four or more not a terminus for transcription. Instead,
T residues within a gene cause premature transcription continued into the intergenic
termination [185, 186]. Deletion of the spacer, initially a further 235 bp to t 2 .
T clusters from the 3' -flanking regions However, t2 did not cause polymerase
causes readthrough beyond the normal release either; rather it formed a boundary
3' -terminus. Similar sequence motifs occur between relatively stable and very unstable
at the 3' -ends of other class III genes, transcript. Transcription continued from t 2
however yeast RNA polymerase may across the intergenic spacer to t3 just 215 bp
require longer runs of Ts because the re- upstream of the initiation site for the next
cognition sequence of most of its 5S RNA tandemly arranged rRNA gene and about
genes consist of 29 T residues. 60 bp from its promoter. The transcript
Termination in vitro is stimulated by a between t 2 and t 3 was rapidly degraded and
highly conserved, 50 kDa nuclear phos- was only detected by run-off transcription or
phoprotein called La. Sera from patients electron microscopy. Thus, t2 is a processing
with autoimmune disease often carry anti- site and it defines the 3' -end of the largest
bodies to La and cell extracts depleted of relatively stable precursor, 40S pre-RNA.
the protein by the antisera lose the ability to The true terminator, t 3 , is a 12 bp element
transcribe class III genes [187, 188]. Purified that is conserved between different am-
La protein restores transcriptional activity phibia [193]. Within the 12 bp is a 7 bp
to these extracts and the protein binds to sequence, GACTTGC, that is also present
the nascent polymerase III transcripts. in t2 . In fact, t 2 can be converted into a
Specifically, it binds to the U-rich 3'-ends of tTlike terminator by a single base change
the RNAs that are created by transcription downstream of the GACTTGC box [194].
of the oligo(T) termination signal [189]. McStay and Reeder [195] have concluded
Gottleib and Steitz [190] have shown that that t 3 and the adjacent promoter of the
La not only binds to the nascent RNA next transcription unit act as one inter-
but facilitates termination. Furthermore, dependent complex bringing about the
Bachmann et al. [191] have demonstrated termination of one transcription unit and
that it is a nucleic acid-dependent A TPase the initiation of the next. They have also
that is capable of using phosphodiester detected a DNA-binding protein that
366 RNA biosynthesis

interacts with the t3 sequence and is required on the initiation of the next rRNA gene.
for termination [196]. The possible importance of this in initiation
Termination elements have also been is discussed in section 9.4.3.
identified in mammalian rONA, particularly
that of the mouse where a repetitive
18 bp motif, known as the Sal box 9.6 DOES THE NUCLEOSKELETON
(AGGTCGACCAGA/TT/ANTCCG) con- PLAY A ROLE IN TRANSCRIPTION?
stitutes the termination signal (Fig. 9.10).
RNA polymerase I specifically terminates Studies with in vitro transcription systems
565 nucleotides downstream of the 3' -end and solubilized RNA polymerase argue that
of 28S RNA and 11 bp upstream of the first transcription is initiated, as described in the
termination signal [197, 198). The 3' -end preceding sections of this chapter, by a
of the transcript is then trimmed by 10 diffusible enzyme binding to a promoter
nucleotides to produce the 3' -terminus of and then moving along the DNA. Cook
the first relatively stable precursor, 45S [202] and others have pointed out that the
RNA [199]. Termination again requires the evidence for this concept of transcription
binding of a specific factor (TTF-1) to the comes from extracts and nuclear fractions
Sal box sequence [200]. The protein is the preparation of which required the use
obviously strongly conserved as it will of hypotonic salt concentrations. The
function in species as divergent as yeast aggregation of chromatin components
and mouse but it has no effect on termin- precludes the use of isotonic or hypertonic
ation by RNA polymerases II and III. solutions under most circumstances but
However, it should be emphasized that, preparations that do employ them suggest
in contrast to the prokaryotic rho factor that active RNA polymerase is associated
and to La protein in termination by RNA with the nucleoskeleton and is not freely
polymer:1se III, the factors so far identified diffusible (reviewed in [202]).
for termination by RNA polymerase I The structure and function of the nucleo-
interact with the DNA not the nascent skeleton is controversial and various struc-
RNA. Parallel studies of the termination tures that can be isolated (nuclear matrix,
of human rONA transcription have iden- nuclear scaffold, nucleoid cages) may be
tified a shorter, 11 bp, recognition motif derived from it to a greater or lesser extent.
(GGGTCGACCAG) that corresponds to Thus, the nuclear matrix is the remnant
the proximal part of the mouse terminator that remains when nuclei are sequentially
[201]. The protein that binds to this element extracted with high salt concentrations (2M
exhibits different electrophoretic mobility NaCI) and non-ionic detergent (1% Triton)
but is otherwise very similar to the mouse and are then treated with DNase and some-
protein [201]. times with RNase. When viewed by electron
As in Xenopus, mammalian rONA microscopy, such samples retain the overall
intergenic spacers contain multiple ter- outline of nuclei but are composed entirely
minator sequences including copies close to of a network of thin proteinaceous fibres
the initiation site for the next tandemly together with residual nucleolar structures,
arranged transcription unit. These appear nuclear pores and connecting lamina.
to have two functions. They terminate Accumulating but conflicting data suggest
transcripts that have initiated in the inter- that the nucleoskeleton may be intimately
genic spacer and they exert a positive effect associated with both transcriptional and
The transcription of genes 367

post-transcriptional processes [203]. DNA [212-214] or that they flank specific re-
appears to be organized into a series of gulatory elements [215, 216]. Sippel and
supercoiled loops anchored to the matrix coworkers [217] have shown that when
and, when the loops are cleaved with endo- a reporter gene is flanked by a scaffold
nucleases, the DNA sequences that remain attachment site of the lysozyme gene, its
attached to the matrix can be analysed. In expression in stably transfected cells is
this way the active ovalbumin [204, 205], significantly elevated and is independent
conalbumin [204], globin [206, 207] and of chromosomal position.
vitellogenin genes [208] have all been shown Perhaps the most radical suggestion
to be preferentially associated with the arising from these studies is a model of
matrix. The data are not, however, uni- transcription in which it is suggested that
versally accepted. Kirov et al. [207] have the template moves past a polymerase
suggested that the apparent association of molecule anchored to the nucleoskeleton.
active genes with the matrix may be an The skeleton is thus seen as the active site of
artifact created by the association of active transcription [202]. At best, the evidence to
genes with proteins that are resistant to the support the model is circumstantial but it
high-salt methods of preparing the matrix. does draw attention to uncertainties in the
When DNase I is used in the absence of accepted concepts. The nuclear matrix has
high salt no such association is observed. also been implicated as the site for the
Mirkovitch et al. [209], also using a low-salt splicing of pre-mRNA and for the transport
extraction method, have shown that histone of mRNA to the cytoplasm (section 11.3.1).
gene clusters are attached to the matrix by Similar arguments to those discussed
an AT-rich region in the spacer between the above centre on whether, in the mitotic
clusters. They report, however, that the chromosome, potentially active genes are
attachment is independent of transcription. associated with the so-called chromosomal
Conversely, Cook and colleagues [210, 211] scaffold (e.g. [218]).
use gentle isotonic methods to make prep-
arations of nucleoskeletons and find that
RNA polymerase, nascent transcripts and 9.7 THE TRANSCRIPTION OF
the genes being transcribed are closely MITOCHONDRIAL AND
associated with it. Their method avoids the CHLOROPLAST GENES
problems of aggregation by encapsulating
cells in agarose microbeads. The beads can The genomes of subcellular organelles
then be extracted with physiological buffers are expressed by their own transcriptional
containing non-ionic detergent so that the machinery that includes specific RNA poly-
cytoplasmic proteins and most RNA diffuses merases. Initially, the characterization of
out to leave encapsulated chromatin sur- these systems lagged well behind those of
rounded by the nucleoskeleton. Transcrip- nuclear transcription because of the low
tional rates assayed in these preparations amounts of the enzymes and the lack of
are twice those of conventional nuclei. specific assays for initiation. However, the
Several groups have more precisely located enzymes have now been purified from
matrix-associated regions (MARs) or several sources and the triphosphates at
scaffold-associated regions (SARs) and the 5'-termini of their transcripts can be
have shown that they coincide with the specifically labelled by the nuclear mRNA-
boundaries of DNase sensitive gene domains capping enzyme, guanyltransferase.
368 RNA biosynthesis

9. 7.1 Mitochondrial transcription and CSB-III that are conserved in vertebrate

mitochondrial DNAs [221].
RNA polymerases have been purified and Other vertebrate species (mouse and
characterized from the mitochondria of Xenopus) appear to have mitochondrial
man, Xenopus and yeast (reviewed in [219]). promoters of similar organization to that
The amphibian and yeast enzymes consist of of man. In yeast, however, there are dif-
a core enzyme and a specificity factor that ferences that reflect the less compact
confers promoter specificity. The human genome. All but one of the tRNA genes
enzyme is similar except that the core are on the same strand in the mitochondrial
enzyme alone exhibits some promoter DNA of the yeast, S. cerevisiae, and some
specificity but requires a factor called mitTF of the genes possess their own promoter
for efficient transcription. The human whereas others are expressed from shared
promoters consist of two components; a promoters. Each promoter consists of a
core promoter and an upstream region. The block of 11 nucleotides with the sequence
factor mitTF binds to the upstream region A/TTATATAAGTA. Transcription begins
and in so doing is presumed to increase the at the last A (position +1) of the sequence.
efficiency with which the core enzyme The core enzyme reacts weakly with the
interacts with the promoter [220, 221]. core element or any other DNA and the
As described in section 8.5.1, vertebrate 43 kDa specificity factor does not interact
mitochondrial genomes are very compact with DNA [222]. However, the two com-
and almost lack the intergenic spacers that ponents co-operate together to form an
would normally contain promoters. In fact, initiation complex [223]. The 11 nucleotide
the whole genome is divergently transcribed promoter sequence also occurs in the DNA
from a single region that includes two replication origin so it seems likely that
independent promoters for leftward and in yeast, as in vertebrates, mitochondrial
rightward transcription (most genes are RNA polymerase may play a role in DNA
encoded on the so-called heavy DNA strand synthesis [219, 221]. The yeast promoter
with just a few tRNA genes on the light does not appear to be strongly conserved in
strand). Furthermore, the promoters are other fungi [224], and neither the nature nor
closely associated with the primary origin the number of mitochondrial promoters is
of DNA replication and are within the D well characterized in higher plants [225].
loop in which a short nascent DNA strand
represents the resting stage of DNA re-
plication (section 6.11.4). It has been 9. 7.2 Chloroplast transcription
suggested that vertebrate mitochondrial
RNA polymerases serve a dual function Chloroplast gene arrangement and ex-
in transcription and replication. Thus, pression has much in common with that
DNA synthesis is seen as initiating with of prokaryotes (reviewed in [226-228]).
the synthesis of a primer RNA at one of Clusters of genes in some cases exhibit
the transcriptional promoters. The switch homology with gene sets in the cyano-
from RNA to DNA synthesis is then be- bacterial genome from which they are
lieved to occur within a 90 nucleotide believed to have evolved. Most of the
region that encompasses three previously genes in these clusters are co-transcribed
identified sequence blocks; CSB-I, CSB-11 into polycistronic mRNAs from promoters
Transcription of DNA viruses 369

that appear to be homologous with the moters share a strongly conserved 23 bp

prokaryotic -10 and -35 motifs. Many of consensus sequence, located at -17 to +6
the promoters can in fact be recognized by with respect to the transcription start site
E. coli RNA polymerase. However, some and for which T7 polymerase has a stringent
chloroplast genes do not have typical pro- specificity [234]. Class III promoters have a
karyotic promoters and there is evidence second conserved motif in the region -22
that chloroplasts may have more than one to -18 [235].
RNA polymerase. A polymerase that is The 17 enzyme consists of a single poly-
isolated as a transcriptionally active DNA- peptide of 98 000 kDa. Transcription is
protein complex, preferentially synthesizes exclusively from promoters on the r stand of
chloroplast rRNA and may be a single the viral DNA and occurs at a very fast rate
polypeptide [229, 230]. Other preparations of 200 nucleotides per second. It selectively
appear to be multi-subunit enzymes and transcribes DNA that is linked to the 23 bp
may specifically transcribe the tRNA and promoter, binds to one face of the DNA
mRNA genes of chloroplasts [231]. It is [269] and all transcripts start with the
possible, however, that these apparent sequence pppGGG [236]. Bacteriophage T3
differences will prove to be artifacts of the RNA polymerase is closely related to that
procedures used and the impurity of the of 17 (82% amino acid identity). It is,
preparations [228]. however, specific for its own promoter motif
which is also a 23 bp sequence but differs
from that of 17 particularly in the region
-10 to -12 [237].
9.8 TRANSCRIPTION OF DNA
Bacteriophage N4 does not rely on the
VIRUSES
host enzyme for early gene transcription
but carries its own, very large (Mr 350000)
9.8.1 Prokaryotic DNA viruses single polypeptide, RNA polymerase within
the bacteriophage particle.
Some bacteriophage redirect the RNA
polymerase of their host to transcribe the
phage genome (sections 10.1.6 and 10.3.2). 9.8.2 Eukaryotic DNA viruses
Others, at least in part, employ their own
enzymes (for a general review of these viral Many of the double-stranded DNA viruses
RNA polymerases, see [232]). that infect animal cells, including SV40,
The RNA polymerase of bacteriophage polyoma, papilloma, adenovirus and Epstein
17 and its relatives (T3, SP6, gh-1) is en- Barr virus (EBV), rely on host RNA
coded by one of the so-called early genes of polymerases to express their genetic pro-
the virus and is transcribed by host RNA gramme. For instance, the genes of EBV
polymerase early in the infective cycle virus are expressed from 24 promoters by
(reviewed in [233]). The transcript is trans- RNA polymerase II [238] and from two
lated by the host's protein-synthesizing promoters by RNA polymerase III [239].
system and viral polymerase is then re- The genomes of these viruses include
sponsible for the transcription of the middle enhancer elements and they encode re-
and late viral genes from class II and class gulatory proteins that allow them to hijack
III promoters, respectively. These pro- their host's transcriptional machinery; often
370 RNA biosynthesis

ensuring that their genes are expressed [243]. The factor consists of two subunits
in multi-tiered cascades. The regulatory of 73 and 82 kDa of which the smaller has
mechanisms that control these processes homology to DNA helicase whereas the
in SV40 and adenovirus are discussed in larger has the leucine zipper and zinc-finger
sections 10.4.4 and 10.4.8, respectively. domains that are associated with many
Relatively few eukaryotic DNA viruses transcriptional factors (section 10.5).
employ viral proteins for replication and Footprinting experiments (section A.9.2)
transcription but the poxviruses are ex- show that the factor interacts with the -13
ceptions; presumably because they replicate to -28 A-rich promoter sequence, A 5TGA8 ,
in the cytoplasm of their host cells. They that specifies the initiation of early genes.
are large viruses. Thus, vaccinia virus It also interacts with the region +7 to
(Copenhagen strain) has a genome of + 10. When interacting with the factor the
191636 bp that includes 198 open reading promoter assumes the conformation of bent
frames of at least 60 amino acids (vaccinia DNA and the major groove widens in the
virus transcription is reviewed in [240] but region of the initiation site. ATP destabilizes
many of the most recent findings described the initiation-factor-polymerase complex
below are sited in a review of a symposium and it has been suggested that the hydrolysis
that was devoted to poxviruses [241 ]). The of ATP releases the complex from the
enzymes encoded in the vaccinia genome promoter and allows the polymerase to
include those required for transcription initiate elongation (from [241]).
(RNA polymerase, poly(A) polymerase and Termination of the early genes occurs
capping enzyme) and those needed for at the sequence TITITNT. It requires
replication (DNA polymerase, DNA ligase, another two-subunit protein which allows
type I topoisomerase, thymidine kinase and the transcriptional complex to recognize the
thymidylate kinase). U 5NU transcript of the termination motif.
When the virion is assembled, the en- Termination is induced approximately 50
zymes required for early gene expression nucleotides further downstream. The ter-
are encapsulated in the core. The uncoating mination factor also functions as a capping
process that follows infection liberates these enzyme; its large subunit possesses tri-
proteins, transcription commences and phosphatase and guanyl transferase activity
results in the production of capped, poly- whereas the small subunit possesses methyl
adenylated mRNAs that are translated on transferase activity [244, 245].
host cell ribosomes [240]. Vaccinia RNA Among the proteins synthesized from the
polymerase has been purified and charac- expression of the early genes is an inter-
terized. It has a Mr of 425000, comprises mediate transcription factor (viTF). It
seven subunits, is resistant -to a-amanitin confers specificity on the viral RNA poly-
and, in vitro, is dependent on Mn 2+ ions for merase for the intermediate gene promoters
activity [242]. The two largest subunits show and these are expressed after the early
sequence homology with those of eukaryotic proteins induce further uncoating of the
RNA polymerases and one small subunit viral DNA. Similarly, the genes transcribed
exhibits homology with the elongation factor in this intermediate class include those
S-11 [241]. The polymerase does not of itself encoding three late gene transcription
possess promoter specificity and recognition factors (vLTFs). They cause the polymerase
of the early gene promoters depends on to recognize the promoters of the so-called
vaccinia early transcription factor (vETF) late genes [240, 241].
Replication of RNA viruses 371

9.9 THE REPLICATION OF RNA and II. They appear to have a stabilizing
VIRUSES BY RNA-DEPENDENT RNA role whereas S1 and host factor probably
POLYMERASE (REPLICASE) function in the binding of replicase to QP
RNA.
9.9.1 RNA bacteriophages

The RNA bacteriophage R17, Qp, MS2 and 9.9.2 Eukaryotic RNA viruses
f2 have 3600-4500 nucleotide, single-
stranded RNA genomes which function (a) Picornaviruses (class IV)
both as a mRNA from which viral proteins
are translated, and as a template for a RNA- These have single-stranded RNA genomes
dependent RNA polymerase (replicase). which can function as mRNAs ( + strands).
Part of the replicase is translated from the The most thoroughly studied example is
infective ( +) strand of the viral RNA which poliovirus, the 7.5 kb genome of which is
it then copies into (-) strands. These are a polyadenylated mRNA linked at its
then also copied to produce more ( +) 5'-terminus to a 22 amino acid peptide,
strands for packaging into progeny infective VPg. The genome is translated by host
bacteriophage. ribosomes into a giant polyprotein. This is
Replicase transcribes RNA from its then cleaved by proteases into a number of
3'-end, initiating a new RNA chain with a proteins including the four viral capsid
5' -GTP and continuing its synthesis in a proteins, part of the replicase and a number
5' ~ 3' direction. It therefore moves along of other products including VPg.
the viral genome in the opposite direction Polio virus RNA polymerase (replicase)
to the ribosomes that are reading it as a has been purified from infected cells and
mRNA. In vitro, a RNA primer can replace from E. coli expressing the protein from
GTP in initiating synthesis. The purification recombinant plasmids. It is a 53 000 kDa
and properties of QP replicase have been protein that is unable to initiate RNA
reviewed [246] and the functional domains synthesis without either an oligonucleotide
of the enzyme have been mapped [247]. The primer or· factors produced by the host
enzyme consists of four subunits of which cell. The role of these factors in viral RNA
subunit II is encoded by the viral genome replication is controversial [248] as is that of
whereas the others are normally part of the VPg ([249] and references therein).
host's protein-synthesizing machinery. They There are two main models for the re-
include the ribosomal protein S1 and the plication of the viral RNA (Fig. 9.11). In the
elongation factors EF-Tu and EF-Ts (the first of these, VPg, or VPg linked to two
normal function of these proteins is de- uridine monophosphates in the form of
scribed in chapter 12). A hexameric protein VPg-pUpU, is postulated to prime RNA
called host factor is also required for QP synthesis [250, 251]. Baltimore [252],
replication. The role that proteins normally suggests the uridylylated VPg hybridizes to
involved in protein synthesis could play the 3'-poly(A) tail of the ( +) strand RNA
in RNA transcription is of considerable and is then elongated by the replicase. The
interest, although incompletely understood. (-) strand so produced is copied by the
It is discussed in section 12.10.2. EF-Tu and replicase after VPg-pUpU has hybridized
EF-Ts are required for replication but are to its two 3'-A residues.
only loosely bound to the complex of S1 A second model proposes that a hairpin
372 RNA biosynthesis

VPg---------------- ~

Hybridization
ofVPg-pUpU

VPg --------------- AAAAAAUUU

VPg AAAAAA

'I
UUVPg Hairpin Formation

VPg AAAAA
UUUAA

! '
Elongation Elongation

VPg AAAAA
UUUAA

VPg AAAAAA
UUUUUUVPg
I
Covalent attachment
of VPg

~
VPg AAAA AA
UUUVPgA
Strand separation
and polyadenylation

/.~-;, nd polyadenylation

VPg AAAAAA

+
AAAAAA VPg

Fig. 9.11 Two models of polio virus replication.

Replication of RNA viruses 373

forms at the 3' -end of the polio virion RNA, Influenza RNA
possibly as a direct result of uridylylation of U CGUUUUC-------

the poly(A) terminus by terminal uridylyl- m7 GpppXmY - - - - - - - - AI _ _ _ _.,.

Elongation
transferase. The hairpin primes the elonga- mRNA capprimer
tion of the complementary (-)strand RNA.
F'ig. 9.12 The priming of influenza virus RNA
In this model (Fig. 9.11), VPg is thought to
synthesis.
function in the trans-esterification required
to break a phosphodiester bond within the
hairpin. The template ( +) strand and the
product (-) strand then separate leaving a but it is not clear whether subsequent
product with VPg linked to its 5' -terminus transcription is continuous through the five
[249]. genes or whether it results from multiple
initiation events. The mRNAs synthesized
(b) Rhabdoviruses (class V) in vitro are typical of those of eukaryotes
in carrying a methylated 5' -cap and a 3'-
Rhabdoviruses have single-stranded, (-) polyadenylated tail. Virus-specific enzyme
strand RNA genomes that cannot function activities carry out these modifications but
as mRNAs. They must be copied into ( +) for the most part have not been identified
strands before they can be expressed. To with specific proteins. The replicase is also
achieve this, the infective viruses contain responsible for replication, namely the
an RNA-dependent RNA polymerase or copying of complete ( +) strands into (-)
replicase (also known as a transcriptase). strands.
The most thoroughly studied example is
vesicular stomatitis virus (VSV), the single- (c) Myxovirus (class V)
stranded genome of which has an Mr of 4 x
106 • It is tightly associated with 1258 copies These viruses have a segmented, single-
of the nucleocapsid protein N and this stranded RNA genome in seven or more
complex, together with smaller quantities of distinct non-overlapping pieces and with
the replicase (protein L [253, 254]) and the negative polarity. The most-studied example
phosphoprotein NS, constitutes the tran- is influenza. The virus encodes and packages
scribing ribonucleoprotein complex (RNP). its own replicating system. However, viral
The replicase synthesizes five distinct species mRNA synthesis also requires cellular
of mRNA from the RNP complex in vitro mRNA, the 5' -terminal portions of which
and in vivo. These include the RNP proteins are used as primers. The 5' -terminal cap
N, L and NS as well as the virion proteins M (m 7GpppXm - section 11.4) of cellular
and G (in the virion, the RNP is coiled into a mRNA is cleaved by a cap-dependent viral
tight helix associated with the matrix protein endonuclease at a purine residue 10-13
M and the glycoprotein G projects through nucleotides downstream from the cap [256].
the outer lipid bilayer that is acquired from The resulting fragments are weakly bonded
the host). to the viral RNA segments, it is presumed
Much remains to be fully understood of by a single base pair between a 3'-terminal
the transcription and replication of VSV and A of the primer and a 3' -U of the viral RNA
other rhabdovirus (reviewed in [255]). RNA (Fig. 9.12). They then function as a primer
synthesis initiates at the 3' -end of the viral for elongation by the viral transcriptase
RNA with the synthesis of a leader RNA (reviewed in [257, 258]).
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