A reduced level of charged tRNAArgmnm5UCU triggers the wild-type peptidyl-tRNA to frameshift

Abstract

Frameshift mutations can be suppressed by a variety of differently acting external suppressors. The +1 frameshift mutation hisC3072, which has an extra G in a run of Gs, is corrected by the external suppressor mutation sufF44. We have shown that sufF44 and five additional allelic suppressor mutations are located in the gene argU coding for the minor tRNAArgmnm5UCU and alter the secondary and/or tertiary structure of this tRNA. The C61U, G53A, and C32U mutations influence the stability, whereas the C56U, C61U, G53A, and G39A mutations decrease the arginylation of tRNAArgmnm5UCU. The T-10C mutant has a base substitution in the -10 consensus sequence of the argU promoter that reduces threefold the synthesis of tRNAArgmnm5UCU . The lower amount of tRNAArgmnm5UCU or impaired arginylation, either independently or in conjunction, results in inefficient reading of the cognate AGA codon that, in turn, induces frameshifts. According to the sequence of the peptide produced from the suppressed -GGG-GAA-AGA- frameshift site, the frameshifting tRNA in the argU mutants is tRNAGlumnm5s2UUC, which decodes the GAA codon located upstream of the AGA arginine codon, and not the mutated tRNAArgmnm5UCU. We propose that an inefficient decoding of the AGA codon by a defective tRNAArgmnm5UCU stalls the ribosome at the A-site codon allowing the wild-type form of peptidyl-tRNAGlumnm5s2UUC to slip forward 1 nucleotide and thereby re-establish the ribosome in the 0-frame. Similar frame-shifting events could be the main cause of various phenotypes associated with environmental or genetically induced changes in the levels of aminoacylated tRNA.

FIGURE 1.

Frameshifting model. A defective cognate tRNA enters the A-site slowly, thereby inducing a stalled ribosome that allows the wild-type cognate peptidyl-tRNA to slip into the +1 frame. The pattern of the nucleotides in the E-, P-, and A-sites corresponds to the triplets found in the wild-type allele of the frameshift mutant.

FIGURE 2.

(A) Cloverleaf structure of tRNA^Arg_mnm⁵UCU with the mutations discussed in this paper indicated. The C61U mutation was isolated in five independent cases, and one of them is the original mutant sufF44; all the other mutations were obtained once. (B) Tertiary structure of tRNA^Phe from yeast (see Kim et al. 1974). The arrows are pointing to the positions of mutated nucleotides.

FIGURE 3.

Relative levels of tRNA^Arg_mnm⁵UCU in wild type and the promoter mutant argU2342 (T-10C). ^aThe bands specific for different deacylated tRNAs were quantified and the r atio between them calculated. The result is the percentage of the level observed in wild type ± standard error. The difference observed between the levels in mutant and in wild type is significant based on a t-test (p = 0.003).

FIGURE 4.

Charging levels of different tRNA^Arg_mnm⁵UCU mutants. Bands corresponding to arginyl- tRNA^Arg_mnm⁵UCU and uncharged tRNA^Arg_mnm⁵UCU are indicated. ^aThe percentage and standard error of arginyl- tRNA^Arg_mnm⁵UCU [100× arginyl-tRNA^Arg_mnm⁵UCU/(arginyl- tRNA^Arg_mnm⁵UCU + uncharged tRNA^Arg_mnm⁵UCU)] are the average of three independent experiments. ^bThe difference in charging observed between the mutant and the wild type is significant based on a t-test (p ≤ 0.03). ^cThe quantification was unreliable because of too high background and diminished amounts of tRNA^Arg_mnm⁵UCU in the T-10C mutant (Fig. 3 ▶). ^dThe double bands of uncharged tRNA in G39A lanes are due to artifacts in this particular gel. The bands migrated as uniform bands in the other two experiments.

FIGURE 5.

Plasmid pUST274 construct used for measuring +1 frameshifting. (A) The malE gene is in the +1 frame relative to the gst gene. In the 3′-terminus of gst, the sequence coding for a PreScission protease recognition site is indicated as a black box; the first stop codon encountered in the 0 frame in malE is marked with an *; 6×His, the six-histidine tag is indicated as a white box. (B) Frameshifting (FS) and termination (Term) products from the wild type and argU2343 (G53C) mutant separated on an SDS gel and were detected by anti-GST antibodies in Western blot analysis.

FIGURE 6.

Various alternatives of fusion proteins produced by slippage by different tRNAs. After PreScission Protease digestion the GST part is cleaved away and the molecular mass of the resulting peptides is analyzed. (I) The peptide part liberated after the cleavage if no frame-shifting occurred; the coding sequence is also presented. The critical AGA codon for R (arginine) is indicated. (II) Fragments of different peptides liberated after the cleavage if different tRNAs were slipping. The amino acid inserted by the frameshifting tRNA and the sequence where this tRNA frameshifts are in bold. The sequence translated and amino acids inserted in the +1 frame are in italic. On the right side are the expected masses of those hypothetical peptides.