Molecular Biology II Translation 1

During translation multiple ribosomes (polysome) move along a single transcript

Nucleotides are read in triplets known as codons. 4nts3 = 64 combinations (enough for 20 AAs)
61 triplets encode for AAs
Nirenberg added synthetic polyribonucleotides (mRNA) to bacterial extracts and showed
that they could make polypeptides.
Crick and Brenner showed that it was a triplet by mutational analysis.
 This shows that the genetic code is degenerate some AAs have more than one codon
There are 3 STOP codons that do not encode AAs UAA / UAG / UGA
All proteins begin with methionine (Met) the start codon is always AUG
Not every AUG is a start codon

Met / Trp are only encoded for by one codon each

Arg / Leu / Ser have 6 codons each

Wobble Hypothesis:
Wobble Interactions
5’ Anti-codon 3’ Codon
Most AAs are represented by codons that differ only in the G C/U
3rd base C G
Cells usually have fewer than 61 tRNAs there is a certain degree of A U
U A/G
play between the 3rd base of the codon and the 1st base of the I (Inosine) A/U/C
anticodon. This allows all triplets to be coded for even if the specific
tRNA is not available. C1 in glycocidic bond is crucial for wobble

The amount of play is defined as ‘wobble’

The 1st base of the anticodon is often referred to as the wobble position
The wobble is possible as non Watson-Crick base pairing can occur (alternative base pairing)

Lecture 22 [Page 1]
Molecular Biology II Translation 1

Codon Usage Phenomenon:

Each organism can recognise each of the 61 AA-encoding codons

Some will be recognised directly if an appropriate tRNA is available
Others will be recognised by tRNA wobbling
Not all codons coding for the same AA are recognised with the same efficiency
There may be fewer tRNAs available for rarely used codons
Highly expressed genes will often use the most ‘popular’ version of the available codons

Fast growing organisms have a larger number of tRNA genes that represent a smaller set of the
possible anti-codons. Fast growers have a more abundant but less diverse set of tRNAs.

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Molecular Biology II Translation 1

The translation machinery in fast growing bacteria has evolved to enlarge the number of tRNAs
and to specialise in the use of a small set of anti-codons.

This codon use is species specific different species will preferentially use certain codons for and
AA they want to encode.

Codon usage had implications in lab work. Heterologous expression of a recombinant protein
involves cloning a gene into a different organism. Sometimes the yield of recombinant protein is
low because the donor and host species have different codon usage. To get around this the host
coding sequence can be modified so that it using the codons utilised most by
E. coli. Or the host can be adapted to use codons that were rarely used previously.

Physico-chemical Hypothesis:

Similar triplets (differing by 1 base) will often encode the same / similar AAs
A correlation exists between ‘hydrophobic ranking’ of the AAs and the hydrophobicity of the anti-
codons themselves. AAs might have been binding directly to codons when genetic code was
established – this binding complementarity might have shaped the evolution of the genetic code.

Evolution of the Genetic Code:

1. Only the AAs that did not depend on other AAs for biosynthesis were encoded
2. Later other AAs that have the early AAs as precursors became available
3. Finally biosynthesis pathways for a number of AAs evolves much later

tRNA – The Adaptor Hypothesis:

tRNA serves as the adaptor to recognise the triplet codon and link it to a specific AA
Specific base pairing occurs between the triplet codon in mRNA and the anti-codon in tRNA
A specific AA is linked covalently to the 3’ end of the tRNA molecule
 Specifically the ACC acceptor stem
tRNA (3D) structure is fairly similar L-shaped with AA at one end anti-codon at the other
RNA is flexible this allows it to self-hybridise at regions of self complimentarity
 Intra-molecular base pairing and base stacking dictate the 3D structure
 The 3D structure is more complex than that of DNA

Lecture 22 [Page 3]
Molecular Biology II Translation 1

Secondary Structure

70-80 nucleotides long

4 self-complementary regions give

rise to a characteristic cloverleaf
structure

The modified
nucleotides will
interact with one another in
different ways leading to non
Watson-Crick base pairing.
There are also some G – U base
pairs. Both of these factors have a
great influence on tertiary (3D)
structure.

Triple
base
pairs can
occur
Tertiary
Structure

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Molecular Biology II Translation 1

CCA Tail Anticodon D Arm

Acceptor Stem Anticodon Arm T (TψC) Arm

Tertiary structure is very important in tRNAs.

tRNA Charging:

Covalent link between AA and tRNA

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Molecular Biology II Translation 1

Aminoacyl tRNA synthetases can recognise subtle differences in AA’s providing specificity, as
there is no base pairing.
There are 2 similar mechanisms for tRNA charging that both begin with the formation of an
AMP-AA (Aminoacyl adenylate) intermediate (1)

The AMP-AA intermediate remains bound to the active site.

Class I aminoacyl-tRNA synthetases

The aminoacyl group is transferred to the tRNA (2)
The aminoacyl group is transferred initially to the 2'-OH group of the 3' end terminal A residue.

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Molecular Biology II Translation 1

A transesterification reaction occurs

moving the AA from the 2' to the 3'
position (3a)

Class II aminoacyl-tRNA synthetases

2b) The aminoacyl group is transferred directly to the 3'-OH group of the 3' end terminal A
residue.

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Molecular Biology II Translation 1

tRNA Synthetases:

Most cells make 20 different structurally diverse aminoacyl-tRNA synthetases (1 / AA)

Some organisms however do not have genes for all 20 AA-tRNA synthetases
 Enzymic reactions are carried out on other AA-tRNAs to get the desired product

Many AA-tRNA transferases can recognise their tRNAs using the anti-codon alone
This is not always possible sometimes the anti-codons for a single AA are very different
 e.g. Serine – 6 codons including AGA / GCU (very different!)
Instead the tRNAs are recognised using segments on the acceptor end and bases elsewhere in
the molecule. This was shown by site-directed mutagenesis studies.

tRNA synthetases approach tRNAs from different angles and make contact with different portions
of the tRNA to gain specificity between structurally similar molecules.

AA-tRNA synthetases must perform task with high accuracy (1 error in 10000)
Most AAs are different to one another so maintaining accuracy is no too difficult
It is harder for the AAs that have very similar size / chemical properties (e.g. Ile & Val)
 Special mechanisms are used to achieve accurate charging
 Ile / Val differ by only a single methyl group and can fit into the same active site
 Val binds instead of Ile 1 in 150 times
Isoleucyl-tRNA synthetase has a second active site, which Ile cannot fit into. The occasional Val
will fit into the site. The mistake is then cleaved away. This proofreading improves the overall
error rate to about 1/3000.

Lecture 22 [Page 8]