Name: M.

Suleman
Roll No: 2015
BS Botany: 7th Semester
Assignment: Plant biochemistry II
Submitted to: Sir Abu Bakar
 Genetic code
• In 1953, James Watson and Francis Crick solved the structure of DNA and identified the base sequence as
the carrier of genetic information. However, the way in which the base sequence of DNA specifies the
amino acid sequences of proteins (the genetic code) remained elusive for another 10 years.
• One of the first questions about the genetic code to be addressed was how many nucleotides are necessary
to specify a single amino acid. The set of nucleotides that encode a single amino acid—the basic unit of the
genetic code—is a codon. Many early investigators recognized that codons must contain a minimum of
three nucleotides.
• Each nucleotide position in mRNA can be occupied by one of four bases: A, G, C, or U. If a codon
consisted of a single nucleotide, only four different codons (A, G, C, and U) would be possible, which is
not enough to encode the 20 different amino acids commonly found in proteins.
• If codons were made up of two nucleotides each (i.e., GU, AC, etc.), there would be 4 × 4 = 16 possible
codons—still not enough to encode all 20 amino acids.
• With three nucleotides per codon, there are 4 × 4 × 4 = 64 possible codons, which is more than enough to
specify 20 different amino acids. Therefore, a triplet code requiring three nucleotides per codon is the most
efficient way to encode all 20 amino acids. Using mutations in bacteriophage, Francis Crick and his
colleagues confirmed in 1961 that the genetic code is indeed a triplet code.
 Breaking the Genetic Code

• When it had been firmly established that the genetic code consists of codons that are three
nucleotides in length, the next step was to determine which groups of three nucleotides
specify which amino acids. Logically, the easiest way to break the code would have been to
determine the base sequence of a piece of RNA, add it to a test tube containing all the
components necessary for translation, and allow it to direct the synthesis of a protein. The
amino acid sequence of the newly synthesized protein could then be determined, and its
sequence could be compared with that of the RNA. Unfortunately, there was no way at that
time to determine the nucleotide sequence of a piece of RNA, so indirect methods were
necessary to break the code.
• The Use of Homopolymers
• The first clues to the genetic code came in 1961, from the work of Marshall Nirenberg and
Johann Heinrich Matthaei. These investigators created synthetic RNAs by using an enzyme
called polynucleotide phosphorylase. Unlike RNA polymerase, polynucleotide
phosphorylase does not require a template; it randomly links together any RNA nucleotides
that happen to be available.
• The first synthetic mRNAs used by Nirenberg and Matthaei were homopolymers, RNA molecules
consisting of a single type of nucleotide. For example, by adding polynucleotide phosphorylase to
a solution of uracil nucleotides, they generated RNA molecules that consisted entirely of uracil
nucleotides and thus contained only UUU codons (Figure 15.8). These poly(U) RNAs were then
added to 20 tubes, each containing the components necessary for translation and all 20 amino
acids. A different amino acid was radioactively labelled in each of the 20 tubes. Radioactive
protein appeared in only one of the tubes—the one containing labelled phenylalanine (see
Figure 15.8). This result showed that the codon UUU specifies the amino acid phenylalanine. The
results of similar experiments using poly(C) and poly(A) RNA demonstrated that CCC encodes
proline and AAA encodes lysine; for technical reasons, the results from poly(G) were
uninterpretable.
The use Of Random Copolymers
• To gain information about additional codons, Nirenberg and his colleagues created synthetic
RNAs containing two or three different bases. Because polynucleotide phosphorylase incorporates
nucleotides randomly, these RNAs contained random mixtures of the bases and are thus called
random copolymers.
• For example, when adenine and cytosine nucleotides are mixed with polynucleotide
phosphorylase, the RNA molecules produced have eight different codons: AAA, AAC, ACC,
ACA, CAA, CCA, CAC, and CCC. These poly(AC) RNAs produced proteins containing six
different amino acids: asparagine, glutamine, histidine, lysine, proline, and threonine.
• Nirenberg and his colleagues could derive information about the base composition of the codons.
These experiments revealed nothing, however, about the codon base sequence; histidine was
clearly encoded by a codon with two Cs and one A, but whether that codon was ACC, CAC, or
CCA was unknown. There were other problems with this method: the theoretical calculations
depended on the random incorporation of bases, which did not always occur, and, because the
genetic code is redundant, sometimes several different codons specify the same amino acid.
The use of ribosome bound tRNAs
To overcome the limitations of random copolymers, Nirenberg and Philip Leder developed another
technique in 1964 that used ribosome-bound tRNAs. They found that a very short sequence of mRNA—
even one consisting of a single codon—would bind to a ribosome.
The codon on the short mRNA would then base pair with the matching anticodon on a transfer RNA that
carried the amino acid specified by the codon (Figure 15.9). Short mRNAs that were bound to ribosomes
were mixed with tRNAs and amino acids, and this mixture was passed through a nitrocellulose filter.
The tRNAs that were paired with the ribosome-bound mRNA stuck to the filter, whereas unbound tRNAs
passed through it. The advantage of this system was that it could be used with very short synthetic mRNA
molecules that could be synthesized with a known sequence.
Nirenberg and Leder synthesized more than 50 short mRNAs with known codons and added them
individually to a mixture of ribosomes and tRNAs. They then isolated the tRNAs that were bound to the
mRNA and ribosomes and determined which amino acids were present on the bound tRNAs. For example,
synthetic RNA with the codon GUU retained a tRNA to which valine was attached, whereas RNAs with
the codons UGU and UUG did not. Using this method, Nirenberg and his colleagues were able to
determine the amino acids encoded by more than 50 codons.
• Other researchers, such as the biochemist Har Gobind Khorana at University of Wisconsin,
extended Nirenberg's experiment by synthesizing artificial mRNAs with more complex sequences.
For instance, in one experiment, Khorana generated a poly-UC (UCUCUCUCUC…) mRNA and
added it to a cell-free system similar to Nirenberg’s.
• The poly-UC mRNA that it was translated into polypeptides with an alternating pattern of serine
and leucine amino acids. These and other results confirmed that the genetic code was based on
triplets, or codons. Today, we know that serine is encoded by the codon UCU, while leucine is
encoded by CUC.
The degeneracy of code
• One amino acid is encoded by three consecutive nucleotides in mRNA, and each nucleotide can have
one of four possible bases (A, G, C, and U) at each nucleotide position, thus permitting 4 3 = 64 possible
codons. Three of these codons are stop codons, specifying the end of translation. Thus, 61 codons,
called sense codons, encode amino acids. Because there are 61 sense codons and only 20 different
amino acids commonly found in proteins, the code contains more information than is needed to specify
the amino acids and is said to be a degenerate code. This expression does not mean that the genetic
code is depraved; degenerate is a term that Francis Crick borrowed from quantum physics, where it
describes multiple physical states that have equivalent meaning.
• The amino acids specified by each codon are given in their three-letter abbreviation. The codons are
written 5′→3′, as they appear in the mRNA. AUG is an initiation codon; UAA, UAG, and UGA are
termination (stop) codons.
• The degeneracy of the genetic code means that amino acids may be specified by more than one codon.
Only tryptophan and methionine are encoded by a single codon. Other amino acids are specified by two
codons, and some, such as leucine, are specified by six different codons. Codons that specify the same
amino acid are said to be synonymous, just as synonymous words are different words that have the
same meaning.
• The Reading Frame and Initiation Codons
• Findings from early studies of the genetic code indicated that the code is
generally nonoverlapping. An overlapping code is one in which a single nucleotide may be
included in more than one codon, as follows:

However, usually each nucleotide is part

of a single codon. A few overlapping
genes are found in viruses, but codons
within the same gene do not overlap,
and the genetic code is generally
considered to be nonoverlapping.
• Generally, AUG codon is the initiating or start codon. The polypeptide chain starts either with
eukaryotes (methionine) or prokaryotes (N- formyl methionine).
• On the other hand, UAG, UAA and UGA are called as termination codons
or stop codons. These are not read by any tRNA molecules and they never
code for any amino acids.
• Non-ambiguous and Universal
• The genetic code is non-ambiguous which means a specific codon will
only code for a particular amino acid. Also, the same genetic code is
seen valid for all the organisms i.e. they are universal
The universality of code
• For many years the genetic code was assumed to be universal, meaning that each codon specifies
the same amino acid in all organisms. We now know that the genetic code is almost, but not
completely, universal; a few exceptions have been found. Most of these exceptions are termination
codons, but there are a few cases in which one sense codon substitutes for another. Most
exceptions are found in mitochondrial genes; a few nonuniversal codons have also been detected
in the nuclear genes of protozoans and in bacterial DNA (Table 15.3).
Wobble hypothesis
• In 1966, Francis Crick developed the wobble hypothesis, which proposed that some nonstandard
pairings of bases could take place at the third position of a codon. For example, a G in the
anticodon may pair with either a C or a U in the third position of the codon (see Table 15.2: note
that inosine in this table is one of the modified base found in tRNAs). The important thing to
remember about wobble is that it allows some tRNAs to pair with more than one codon on an
mRNA; thus from 30 to 50 tRNAs can pair with 61 sense codons. Some codons are synonymous
through wobble.