Lecture 8 -Transcription
p II
Produces toxin that
prevents RNA polyme-
rase from working -->
eukaryotic RNA polymerase
Green is shared between bacteria and eukaryotes
Eu: 12 subunits, Bac: 5 subunits
Transcription in eukaryotes
Lecture Outline: Readings:
1)) Structure of a g
gene Alberts, Ch 6 -
pp.339-358
2) RNA polymerases (just to the end
3) Transcription of protein-
protein of 3’processing
3 processing
coding genes section)
4)) RNA pprocessing
ocess g
2
Recall the more complex eukaryotic gene structure
DNA
primary RNA
transcript <- very rarely seen
5’ RNA cap
PolyA tail
Alberts Figure 6-21
Alberts,
Introns are transcribed but not translated 3
Exons are transcribed, and may or may not be translated
An expanded repertoire of RNAs to generate
m,r,&t code for
protein
Table 6-1 Molecular Biology of the Cell (© Garland Science 2008)
A number of eukaryotic RNA polymerases
required
• Each of these RNAPs is a multi-subunit
protein
• Each is responsbile for transcription of
different RNAs
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structurally similar, and have similar sub-units, but transcribe different genes
Subunits of eukaryotic RNA polymerases
RNAP’s are complex
structures with many
subunits:
1) Some
S subunits
b it are
carboxy terminal
common to all three domain
RNAP s
RNAP’s
2) Some subunits
resemble the
subunits of:
Bacterial
Bacterial RNAPs
RNAP’s
6
Subunits of RNA polymerases
require more proteins called 'general transcription
7 factors'
has to deal with unravelling and coiling
Eukaryotic vs. bacterial RNA polymerases
1) Eukaryotic RNA polymerases require
proteins to help position them at the
promoter, called:
transcription
Transcription factors, similarfactors
to the sigma subunit
2) These factors fulfill a similar role to the
___________
sigma
similar to thesubunit
sigma subunit of the bacterial RNA
polymerases
3) Eukaryotic
E k ti RNA polymerases l need
d tto
deal with ___________________
chromosomal
chromosomal structures
structures, nucleosomes, etc.
8
Gene structure
No introns in prokaryotes, just a eu thing
(translated) (not translated)
multiple
lti l
proteins
polysystronic
Introns removed
monosystronic
Pol A tail
Poly-A
5’ cap
9
Alberts, Figure 6-22a
guanine, methyl group
Transcription of protein-coding genes
1) Promoters
2) Initiation of transcription
3) RNA processing i
10
"upstream":
negative numbers,
+1 Promoters
RNA polymerase starts making mRNA; "downstream" - are transcribed
not transcribed
calls RNAP here! positions RNAP before it
"TFII" is the
transcription factor
for RNAP II
'n' means any
nucleotide "TBP" is TATA
binding protein
helps to position the RNAP for transcription , is NOT transcribed; before start point
1) TATA box: the sequence TATA highly
conserved, found 25-36 bp upstream from
start site for transcription
p
Positions RNAP II *Not every gene has all four
Highly transcribed genes promoters!
Alberts, Figures 6-17 & 6-18 11
Transcription of protein-coding genes
1) Promoters
2) Initiation of transcription
3) RNA processing i
12
Steps in the initiation of transcription
Legend:
transcription factors bind
TBP is subunit of TFIID, in much larger, 9 subunits;
highly transcribed genes, binds 'H' is for helicase
to TATA box opens up DNA strands
requires ATP
binds to BRE element, helps pos. RNAP II
helps separate also
similar to sigma, helps units assemble w/ RNAP II
Transcription
p
initiation
complex
13
Alberts, Figure 6-16
Steps in the initiation of transcription
Legend:
transcription factors bind
abortive transcription: stops and starts
TFIIH comes in and makes it go crazy
fast
14
Alberts, Figure 6-16
Steps in the initiation of transcription
TBP
TBP
1) ____, a subunit of _____, TFIID
TFIID
binds to the TATA
box promoter in the minor groove groove, bending and
distorting the DNA to a shape more suited for RNAP II
2) This attracts other transcription factors factors, which
help to orient and bind ________ RNAP
RNAP II II to the DNA
3) The helicase activity of _____ TFIIH
TFIIH uses ATP to pry
apart DNA strands at transcription start site
phosphorylates
p p y
4) TFIIH also _____________
phosphorylates the CC-terminal
terminal tail
of RNA polymerase II, activating it so that
transcription can begin called "kynase" activity, anything that phosphorylates
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RNA polymerase II C-terminal tail
In RNAP II only the carboxy terminal domain of the
l
largest
t subunit
b it hhas a stretch
t t h off 7 amino
i acids
id th
thatt
is repeated multiple times
Serines get phosphorylated, **** 5th will be phos.ed by TFIIH
1)Repeat = Tyr-Ser-Pro-Thr-Ser-Pro-Ser
26 repeats
2)Yeast enzyme has ___
3)Human enzyme has ___
52 repeats
This region is essential for viability
16
Additional factors required for transcription initiation
activator +1
enhancer calls for act.
to come bind
site to it
Activators help signal
binding of transcription
factors and polymerases
Communicates w/ t. factor
DNA loop and polymerases, tells
them to activate
Calls ATP for the h-m
enzyme and chr-remod
complex
Alberts, Figure 6-19 17
Review Q’s
1) How is RNA polymerase II activated?
Phosphorylation
Ph h l i
Phosphorylation
2) What
Wh t iis phosphorylated?
h h l t d?
Adding phosphate
Adding phosphate groups
groups to fifth Serine onterminal
of the carboxy S (Ser)
group
located on the CTD
3) How many proteins are involved in
initiating eukaryotic transcription?
>100 subunits
>100 subunits
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Transcription of protein-coding genes
1) Promoters
2) Initiation of transcription
3) RNA processing i
19
5’ pre-mRNA capping
Remove
ONE P is removed
phosphate
*phosphotase* opp. of kynase
1) Helps to Cap helps prevent
________
protect
protect RNA exos. from chewing
off nucleotides
Add GMP pyrophosphate
comes off
from *guanyltransferase*
exonucleases
2) Last step is in Add methyl
group to G base
some
______
some caps
Add methyl group
to 2nd ribose
* guanine becomes first ribose
*methyltransferase*
20
Alberts, Figure 6-24
5’ cap structure
5’ to 5’ link
the guanine cap is added backwards!
Alberts, Figure 6-22B
21
RNA factory
Phosphorylation of C-
C
terminal tail of RNA 5th Ser is phosphorylated
Polymerase II results in
binding of: after 25 ns, cap comes in
RNARNA
processing
s.p.s remove introns and
proteins
processing some exons
proteins
Alberts, Figure 6-23
22
RNA processing increases the number of gene products
arrow shows spliced out part
working muscles
don't consciously control
Figure 6-27 Molecular Biology of the Cell (© Garland Science 2008)
The pre-mRNA splicing reaction
soecific sequences in the intron
Two step process:
branches out to allow binding to other parts
catalysed by proteins that help
1)) Adenosine
Adenosine
remove introns _________ attacks the
5’ splice site
2) 3’ of one exon reacts
with 5’ of next exon to
iintron
intron
t
release ______
They merge
Alberts, Figure 6-26a
24
The catalytic mechanism is RNA dependent
2’ OHgroup group
3) __________
2' OH
of the
ribose sugar is not
present in
deoxyribose
4) This group is
necessary for the
formation of the
lariat
______
lariat
structure in
intron splicing
The O on the 2' end attacks another end (5' end of
another) and binds to create the 'lasso shape'
Alberts, Figure 6-26b 25
Sequences
q required
q for intron removal
the branch point,
cannot change forms 2' phosphate bond
cannot change
Y: C/U pyrimidine
R: A/G purine
Alberts, Figure 6-28 26
RNA and Protein
interactions for splicing
snRNA = small nuclear
RNAs
1) snRNP’s
snRNP s =
snRNA’s
snRNAs + proteins+ proteins
__________________
2) Pre
Pre-mRNAmRNA splicing
is performed by the
spliceosome
p
__________
spliceosome
Alberts, Figure 6-29 27
Within the spliceosome
snRNPs "U-"s
Alberts, Figure 6-30c
ATP dependent
bind to many nucleotide sequences
28
When splicing goes bad.....
mutants with snRNPs, spliceosomes can cause many diseases - cystic fibrosis
Result in betathalosimi... needs blood transfusions
Figure 6-35 Molecular Biology of the Cell (© Garland Science 2008)
RNA processing to generate the 3’ end also required
RNAP makes RNA • Consensus sequences
CTD gets phos'd direct cleavage and
polyadenylation of the 3’ end
Transcribed, changed
• These sequences
q are
to RNA encoded by the DNA to be
Proteins transferred to transcribed
RNA
• Specific proteins recognize
these sequences in the RNA
molecule
o ecu e
A's added to protect
• Cleavage and addition of a
the protein chain poly-A 3’ tail result in the
mature mRNA
Figure 6-38 Molecular Biology of the Cell (© Garland Science 2008)
Transcription of the consensus sequences and recruitment of
modifying proteins
CPSF and
CPSF (Cleavage CtsF
and polyadenylation
specificity factor), and CtsF (Cleavage
move
stimulationfrom the CTD
factor) move
to specific sequences
on the RNA
Figure 6-38 (part 1 of 3) Molecular Biology of the Cell (© Garland Science 2008)
RNA is cleaved and polyadenlylated
PAP
•PAP requires
requires nosimply adds
no template,
As to the 3' end of the mRNA
template – tosimply
number of As (up adds
~200) determined
by poly-A binding proteins
A’s
A s to the 3’end3 end of the
mRNA
• number of A’s
A s (up to
~200) determined by
polyA binding proteins
Figure 6-38 (part 2 of 3) Molecular Biology of the Cell (© Garland Science 2008)
Mature mRNA exported from the nucleus to the cytoplasm
mRNA translated in the cytoplasm - site of protein synthesis
mRNA
w/out captranslated inget
and tail, it doesn't the cytoplasm
transported –digested
properly, site oforprotein synthesis
otherwise destroyed
Figure 6-39a Molecular Biology of the Cell (© Garland Science 2008)
THE END
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