9. tRNA may influence the reading frame

7.9 tRNA may influence the reading frame

The reading frame of a messenger usually is invariant. Translation starts at an AUG codon and continues in triplets to a termination codon. Reading takes no notice of sense: insertion or deletion of a base causes a frameshift mutation, in which the reading frame is changed beyond the site of mutation. Ribosomes and tRNAs continue ineluctably in triplets, synthesizing an entirely different series of amino acids.

There are some exceptions to the usual pattern of translation that enable a reading frame with an interruption of some sort Xsuch as a nonsense codon or frameshift Xto be translated into a full-length protein. Recoding events are responsible for making exceptions to the usual rules, and can involve several types of events:

• Suppression involves recognition of a codon by a tRNA that usually would respond to a different codon (as in the examples of nonsense suppression discussed earlier).
  
• Redefinition of the meaning of a codon occurs when an aminoacyl-tRNA is modified (as in the example of the substitution of selenocysteine discussed earlier).
  
• Frameshifting typically involves changing the reading frame by +1 or V1 at a specific site.
  

Frameshift mutations are suppressed by restoring the original reading frame. We have already discussed how this can be achieved by compensating base deletions and insertions within a gene (see 1 Genes are DNA). However, extragenic frameshift suppressors also can be found in the form of tRNAs with aberrant properties.

The simplest type of external frameshift suppressor corrects the reading frame when a mutation has been caused by inserting an additional base within a stretch of identical residues. For example, a G may be inserted in a run of several contiguous G bases. The frameshift suppressor is a tRNA^Gly that has an extra base inserted in its anticodon loop, converting the anticodon from the usual triplet sequence CCC^← to the quadruplet sequence CCCC^← . The suppressor tRNA recognizes a 4-base "codon."

Some frameshift suppressors can recognize more than one 4-base "codon." For example, a bacterial tRNA^Lys suppressor can respond to either AAAA or AAAU, instead of the usual codon AAA. Another suppressor can read any 4-base "codon" with ACC in the first three positions; the next base is irrelevant. In these cases, the alternative bases that are acceptable in the fourth position of the longer "codon" are not related by the usual wobble rules. The suppressor tRNA probably recognizes a 3 base codon, but for some other reason Xmost likely steric hindrance Xthe adjacent base is blocked. This forces one base to be skipped before the next tRNA can find a codon.

Situations in which frameshifting is a normal event are presented by phages and viruses. Such events may affect the continuation or termination of protein synthesis, and result from the intrinsic properties of the mRNA.

In phage MS2, a frameshift causes the ribosome to recognize a termination codon at an early position in its new reading frame. The terminating ribosome then can recognize the initiation codon of the lysis gene, which lies just a few bases farther along. When the ribosome does not terminate, it reads right over the lysis gene initiation codon. So the frameshift-dependent termination event is a prerequisite for initiation of lysis gene expression.

Figure 7.22 A +1 frameshift is required for expression of the tyb gene of the yeast Ty element. The shift occurs at a 7 base sequence at which two Leu codon(s) are followed by a scarce Arg codon.

In retroviruses, translation of the first gene is terminated by a nonsense codon in phase with the reading frame. The second gene lies in a different reading frame, and (in some viruses) is translated by a frameshift that changes into the second reading frame and therefore bypasses the termination codon (see 16 Retroviruses and retroposons). Figure 7.22 illustrates the similar situation of the yeast Ty element, in which the termination codon of tya must be bypassed by a frameshift in order to read the subsequent tyb gene.

Such situations makes the important point that the rare (but predictable) occurrence of "misreading" events can be relied on as a necessary step in natural translation. This is called programmed frameshifitng. It occurs at particular sites at frequencies that are 100 V1000 greater than the rate at which errors are made at nonprogrammed sites (~3 10 ^V5 per codon).

There are two common features in this type of frameshifting:

• A "slippery" sequence allows an aminoacyl-tRNA to pair with its codon and then to move +1 (rare) or V1 base (more common) to pair with a triplet sequence that can also pair with its anticodon.
  
• The ribosome is delayed at the frameshifting site to allow time for the aminoacyl-tRNA to rearrange its pairing. The cause of the delay can be an adjacent codon that requires a scarce aminoacyl-tRNA, a termination codon that is recognized slowly by its release factor, or a structural impediment in mRNA (for example, a "pseudoknot," a particular conformation of RNA) that impedes the ribosome.
  

The frameshifting in Figure 7.22 shows the behavior of a typical slippery sequence. The 7 nucleotide sequence CUUAGGC is usually recognized by Leu-tRNA at CUU followed by Arg-tRNA at AGC. However, the Arg-tRNA is scarce, and when its scarcity results in a delay, the Leu-tRNA slips from the CUU codon to the overlapping UUA triplet. This causes a frameshift, because the next triplet in phase with the new pairing (GGC) is read by Gly-tRNA. Slippage usually occurs in the P site (when the Leu-tRNA actually has become peptidyl-tRNA, carrying the nascent chain.

Slippery events can involve movement in either direction; a V1 frameshift is caused when the tRNA moves backwards, and a +1 frameshift is caused when it moves forwards. In either case, the result is to expose an out-of-phase triplet in the A site for the next aminoacyl-tRNA.

Frameshifting also occurs in some cases in which the codon in the second phase cannot be reached by slipping from the codon in the original phase. One explanation is that a tRNA initially enters and binds to a codon that is out of phase. An alternative is that a tRNA with a normal anticodon occasionally recognizes a 4 base nucleotide sequence instead of a triplet. This mechanism shows the same need to delay the ribosome as frameshifting at slippery sequences.

Frameshifting at a stop codon causes readthrough of the protein. The base on the 3′ side of the stop codon influences the relative frequencies of termination and frameshifting, and thus affects the efficiency of the termination signal. This helps to explain the significance of context on termination (for review see 43, 44).

Reviews
43:	Farabaugh, P. J. (1995). Programmed translational frameshifting. Microbiol. Rev. 60, 103-134.
44:	Gesteland, R. F. and Atkins, J. F. (1996). Recoding: dynamic reprogramming of translation. Ann. Rev. Biochem 65, 741-768.