Berg J.M., Tymoczko J.L., Stryer L. Biochemistry

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the ATP that is cleaved by the aminoacyl-tRNA synthetase before the arrival of the tRNA at the ribosome.

With the peptide bond formed, the peptide chain is now attached to the tRNA in the A site on the 30S subunit while a

change in the interaction with the 50S subunit has placed that tRNA and its peptide in the P site of the large subunit. The

tRNA in the P site of the 30S subunit is now uncharged. For translation to proceed, the mRNA must be moved (or

translo-cated) so that the codon for the next amino acid to be added is in the A site. This translocation takes place

through the action of a protein enzyme called elongation factor G (Section 29.4.3), driven by the hydrolysis of GTP. On

completion of this step, the peptidyl-tRNA is now fully in the P site, and the uncharged initiator tRNA is in the E site

and has been disengaged from the mRNA. On dissociation of the initiator tRNA, the ribosome has returned to its initial

state except that the peptide chain is attached to a different tRNA, the one corresponding to the first codon past the

initiating AUG. Note that the peptide chain remains in the P site on the 50S subunit throughout this cycle, presumably

growing into the tunnel. This cycle is repeated as new aminoacyl-tRNAs move into the A site, allowing the polypeptide

to be elongated indefinitely.

We can now understand why the amino terminus of the initial methionine molecule is modified by the attachment of a

formyl group. Chemical reactivity may have dictated this modification (Figure 29.26). Suppose that the amino-terminus

is not blocked. After the first peptidyl-transfer reaction, a dipeptide is linked to the tRNA in the P site. If a free amino

group is present in the terminal amino acid, this amino group can attack the carbonyl group of the ester linkage to the

tRNA, forming a very stable six-membered ring and terminating translation.

29.3.8. Only the Codon-Anticodon Interactions Determine the Amino Acid That Is

Incorporated

On the basis of the mechanism described in Section 29.3.7, the base-pairing interaction between the anticodon on the

incoming tRNA and the codon in the A site on mRNA determines which amino acid is added to the polypeptide chain.

Does the amino acid attached to the tRNA play any role in this process? This question was answered in the following

way. First, cysteine was attached to its cognate tRNA. The attached cysteine unit was then converted into alanine by

adding Raney nickel to Cys-tRNA

Cys

; the reaction removed the sulfur atom from the cysteine residue without affecting

its linkage to tRNA. Thus, a mischarged aminoacyl-tRNA was produced in which alanine was covalently attached to a

tRNA specific for cysteine.

Does this mischarged tRNA recognize the codon for cysteine or for alanine? The answer came when the tRNA was

added to a cell-free protein-synthesizing system. The template was a random copolymer of U and G in the ratio of 5:1,

which normally incorporates cysteine (encoded by UGU) but not alanine (encoded by GCN). However, alanine was

incorporated into a polypeptide when Ala-tRNA

Cys

was added to the incubation mixture. The same result was obtained

when mRNA for hemoglobin served as the template and [

C]alanyl-tRNA

Cys

was used as the mischarged aminoacyl-

tRNA. The only radioactive tryptic peptide produced was one that normally contained cysteine but not alanine. Thus, the

amino acid in aminoacyl-tRNA does not play a role in selecting a codon.

In recent years, the ability of mischarged tRNAs to transfer their amino acid cargo to a growing polypeptide chain has

been used to synthesize peptides with amino acids not found in proteins incorporated into specific sites in a protein.

Aminoacyl-tRNAs are first linked to these unnatural amino acids by chemical methods. These mischarged aminoacyl-

tRNAs are added to a cell-free protein-synthesizing system along with specially engineered mRNA that contains codons

corresponding to the anticodons of the mischarged aminoacyl-tRNAs in the desired positions. The proteins produced

have unnatural amino acids in the expected positions. More than 100 different unnatural amino acids have been

incorporated in this way. However, only l-amino acids can be used; apparently this stereochemistry is required for

peptide-bond formation to take place.

29.3.9. Some Transfer RNA Molecules Recognize More Than One Codon Because of

Wobble in Base-Pairing

What are the rules that govern the recognition of a codon by the anticodon of a tRNA? A simple hypothesis is that each

of the bases of the codon forms a Watson-Crick type of base pair with a complementary base on the anticodon. The

codon and anticodon would then be lined up in an antiparallel fashion. In the diagram in the margin, the prime denotes

the complementary base. Thus X and X

would be either A and U (or U and A) or G and C (or C and G). According to

this model, a particular anticodon can recognize only one codon.

The facts are otherwise. As found experimentally, some pure tRNA molecules can recognize more than one codon. For

example, the yeast alanyl-tRNA binds to three codons: GCU, GCC, and GCA. The first two bases of these codons are

the same, whereas the third is different. Could it be that recognition of the third base of a codon is sometimes less

discriminating than recognition of the other two? The pattern of degeneracy of the genetic code indicates that this might

be so. XYU and XYC always encode the same amino acid; XYA and XYG usually do. Francis Crick surmised from

these data that the steric criteria might be less stringent for pairing of the third base than for the other two. Models of

various base pairs were built to determine which ones are similar to the standard A · U and G · C base pairs with regard

to the distance and angle between the glycosidic bonds. Inosine was included in this study because it appeared in several

anticodons. With the assumption of some steric freedom ("wobble") in the pairing of the third base of the codon, the

combinations shown in Table 29.3 seemed plausible.

The wobble hypothesis is now firmly established. The anticodons of tRNAs of known sequence bind to the codons

predicted by this hypothesis. For example, the anticodon of yeast alanyl-tRNA is IGC. This tRNA recognizes the codons

GCU, GCC, and GCA. Recall that, by convention, nucleotide sequences are written in the 5

3 direction unless

otherwise noted. Hence, I (the 5

base of this anticodon) pairs with U, C, or A (the 3 base of the codon), as predicted.

Two generalizations concerning the codon-anticodon interaction can be made:

1. The first two bases of a codon pair in the standard way. Recognition is precise. Hence, codons that differ in either of

their first two bases must be recognized by different tRNAs. For example, both UUA and CUA encode leucine but are

read by different tRNAs.

2. The first base of an anticodon determines whether a particular tRNA molecule reads one, two, or three kinds of

codons: C or A (one codon), U or G (two codons), or I (three codons). Thus, part of the degeneracy of the genetic code

arises from imprecision (wobble) in the pairing of the third base of the codon with the first base of the anticodon. We see

here a strong reason for the frequent appearance of inosine, one of the unusual nucleosides, in anticodons. Inosine

maximizes the number of codons that can be read by a particular tRNA molecule. The inosines in tRNA are formed by

deamination of adenosine after synthesis of the primary transcript.

Why is wobble tolerated in the third position of the codon but not in the first two? The 30S subunit has two adenine

bases (A1492 and A1493 in the 16S RNA) that form hydrogen bonds on the minor-groove side of the codon-anticodon

duplex. These interactions serve to check whether Watson-Crick base pairs are present in the first two positions of the

codon- anticodon duplex. No such inspection device is present for the third position so more-varied base pairs are

tolerated. This mechanism for ensuring fidelity is analogous to the minor-groove interactions utilized by DNA

polymerase for a similar purpose (Section 27.2.3). Thus, the ribosome plays an active role in decoding the codon-

anticodon interactions.