Special peptidyl-tRNA molecules can promote translational frameshifting without slippage.

Abstract

Recently we described an unusual programmed +1 frameshift event in yeast retrotransposon Ty3.Frameshifting depends on the presence of peptidyl-tRNA " on the GCG codon in the ribosomal P site and on a translational pause stimulated by the slowly decoded AGU codon. Frameshifting occurs on the sequence GCG-AGU-U by out-of-frame binding of a valyl-tRNA to GUU without slippage of peptidyl-tRNAV&. This mechanism challenges the conventional understanding that frameshift efficiency must correlate with the ability of mRNA-bound tRNA to slip between cognate or near-cognate codons. Though frameshifting does not require slippery tRNAs, it does require special peptidyl-tRNAs. We show that overproducing a second isoacceptor whose anticodon had been changed to CGC eliminated frameshifting; peptidyl-tRNAVZ' must have a special capacity to induce +1 frameshifting in the adjacent ribosomal A site. In order to identify other special peptidyl-tRNAs, we tested the ability of each of the other 63 codons to replace GCG in the P site. We found no correlation between the ability to stimulate + 1 frameshifting and the ability of the cognate tRNA to slip on the mRNA-several codons predicted to slip efficiently do not stimulate frameshifting, while several predicted not to slip do stimulate frameshifting. By inducing a severe translational pause, we identified eight tRNAs capable of inducing measurable +1 frameshifting, only four of which are predicted to slip on the mRNA. We conclude that in Saccharomyces cerevisiae, special peptidyl-tRNAs can induce frameshifting dependent on some characteristic(s) other than the ability to slip on the mRNA.Protein synthesis produces a low frequency of errors: both missense errors and processivity errors (those leading to premature termination). However, spontaneous frameshift errors are extremely infrequent (34). This means that the translational machinery must much more efficiently avoid changes in frame than it does other errors. Some genes have evolved sequences which manipulate this machinery to allow very high levels of frameshifting, with efficiencies from a few to nearly 100% (2, 22, 28). All such sequences, termed programmed frameshift sites, consist of two elements: the actual site at which the frame shifts, termed the recoding site (22), and sequences which increase the probability that the ribosome will slip, termed stimulators (2).In all but one case, the recoding site is a sequence which allows slippage of one or more tRNAs between cognate or near-cognate codons and thus is termed a slippery site. Frameshifts occur in either the upstream, or leftward, direction (e.g., -1 frameshift) or in the downstream, or rightward direction (e.g., +1 frameshifting). In -1 simultaneous slippage frameshifting, common in retroviruses but found in other viruses and one chromosomal gene, the slippery site is a heptamer of the form X-XXY-YYZ. Frameshifting occurs by the simultaneous slippage of two tRNAs from XXY-YYZ to 46). Mutational changes which decrease the likelihood of the slip tend to decrease or elimi...

Nonsense-mediated decay mutants do not affect programmed −1 frameshifting

“…The lacZ-luc dual-reporter system sensitively reported the effect of several trans-acting factors on a variety of recoding sites+ Some effects were quite strong; forcing near-cognate recognition of the AGG pause codon caused essentially all ribosomes that encountered the Ty1 programmed ϩ1 frameshift site to shift frames+ These results confirmed those obtained using a lacZ single-reporter system (Vimaladithan & Farabaugh, 1994)+ Other results were nearly as dramatic, such as the approximately four-to sixfold increase in nonsense codon readthrough in a [PSI ϩ ] strain+ In each of these cases we can explain the large increase as the result of restricting normal decoding in the A site+ The nearcognate tRNA Arg UCU probably reads an AGG codon very inefficiently because the wobble uridine is modified to recognize nucleotides other than A very poorly (as discussed by Vimaladithan & Farabaugh, 1994)+ Similarly, in a [PSI ϩ ] strain the availability of eRF is restricted because an inactive complex sequesters it, making it unavailable to promote termination+ Overexpression of FIGURE 4. Wild-type strain HFY1200 and the congenic HFY870 (⌬upf1), HFY1300 (⌬upf2), and HFY863 (⌬upf3 ) strains were transformed with pACTMV (UAG), pACTGA, pACTAA, or pACTQ+ Readthrough efficiency is expressed as in Figure 2+ FIGURE 5.…”

“…Schematic of the lacZ-luc reporter system+ The reporter consists of tandem lacZ and luc genes separated by a short linker+ The ends of the linker are NheI and Bcl I restriction sites that can be used to insert oligonucleotides bearing recoding sites+ The figure shows the introduction of an oligonucleotide carrying the TMV termination readthrough site+ When inserting such an oligonucleotide, the sequence of the Bcl I site was changed to eliminate an in-frame TGA termination codon that would otherwise block continued translation into luc+ Another type of programmed frameshift site similar to shifty stops are those that use a poorly recognized sense codon to stimulate ϩ1 frameshifting (Belcourt & Farabaugh, 1990;Peter et al+, 1992;Farabaugh et al+, 1993)+ At such sites the slow recognition of an in-frame sense codon allows the peptidyl-tRNA bound to the immediately upstream codon to stimulate a ϩ1 shift in frames+ The recoding sites derived from the yeast retrotransposon Ty1 contain such a site stimulated by the slow recognition of an AGG codon by the rare tRNA Arg CCU (Belcourt & Farabaugh, 1990)+ Using a single-gene reporter system, we have previously shown that deleting the single gene encoding tRNA Arg CCU (⌬hsx1) to force decoding by the near-cognate tRNA greatly stimulates ϩ1 frameshifting (Kawakami et al+, 1993;Vimaladithan & Farabaugh, 1994)+ Using the dualgene reporter system we tested the effect of ⌬hsx1 on frameshifting on the Ty1 programmed frameshift site, CUU-AGG-C+ In accord with our previous results, ⌬hsx1 strongly stimulates ϩ1 frameshifting+ Remarkably, the value we determine for frameshifting in the Figure 2+ strain KK240 (⌬hsx1) is 100%+ This means that essentially all ribosomes that encounter the frameshift signal shift into the ϩ1 reading frame, whereas in the congenic strain KK242 (HSX1), which has a normal concentration of tRNA Arg CCU , frameshifting as expected is much lower, 30%+ The fact that the system measures the genes both upstream and downstream of the frameshift site eliminates the uncertainty about the meaning of the high level of expression that we had from our previous study using the single-gene reporter+ Apparently in this genetic background, the ribosome is virtually incapable of properly decoding CUU-AGG-C+…”

Lack of Pseudouridine 38/39 in the Anticodon Arm of Yeast Cytoplasmic tRNA Decreases in Vivo Recoding Efficiency

“…they consist of a frameshift-inducing codon followed by an in-frame termination opal codon UGA. The frameshiftinducing codons used in these constructs (CUU for leucine, CCG for proline, and GCG for alanine) were chosen because they promote the highest level of ϩ1 frameshifting compared with other codons in yeast (49). According to previous work, the frameshift-inducing codons CUU, CCG, and GCG will be read by tRNA UAG Leu , tRNA U*GG Pro , and tRNA IGC Ala , respectively (43,50).…”

Ribosomal Frameshift Signals in Viral Genomes

“…The scarcity of the aminoacyl-tRNA for the Ty3 is limiting, indicating that a pause is required. Vimaladithan and Farabaugh (1994) showed that increasing the abundance of the tRNA decreased frameshifting and removal of the tRNA enhanced frameshifting. The nature of the codon:anticodon interaction at the P-site is proposed to leave the first base of the A-site codon unpaired at tRNA accommodation (Sundararajan et al, 1999).…”