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Comparative Study
. 2001 Sep 3;20(17):4863-73.
doi: 10.1093/emboj/20.17.4863.

Improvement of reading frame maintenance is a common function for several tRNA modifications

J Urbonavicius  1 , Q Qian, J M Durand, T G Hagervall, G R Björk
Affiliations
Comparative Study

Improvement of reading frame maintenance is a common function for several tRNA modifications

J Urbonavicius et al. EMBO J. .

Abstract

Transfer RNAs from all organisms contain many modified nucleosides. Their vastly different chemical structures, their presence in different tRNAs, their occurrence in different locations in tRNA and their influence on different reactions in which tRNA participates suggest that each modified nucleoside may have its own specific function. However, since the frequency of frameshifting in several different mutants [mnmA, mnmE, tgt, truA (hisT), trmD, miaA, miaB and miaE] defective in tRNA modification was higher compared with the corresponding wild-type controls, these modifications have a common function: they all improve reading frame maintenance. Frameshifting occurs by peptidyl-tRNA slippage, which is influenced by the hypomodified tRNA in two ways: (i) a hypomodified tRNA in the ternary complex may decrease the rate by which the complex is recruited to the A-site and thereby increasing peptidyl-tRNA slippage; or (ii) a hypomodified peptidyl-tRNA may be more prone to slip than its fully modified counterpart. We propose that the improvement of reading frame maintenance has been and is the major selective factor for the emergence of new modified nucleosides.

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Figures

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Fig. 1. A dual-error model for frameshifting. (A) Hypomodified cognate tRNA is defective in the aa-tRNA selection step, thereby allowing a wild-type near-cognate tRNA to be accepted instead at the A-site. After a normal three nucleotide translocation, the near-cognate tRNA slips into either –1 or +1 frame, depending on the sequence of the mRNA. (B) The hypomodified cognate tRNA is very slow in entering the A-site, inducing a pause and thereby allowing the wild-type cognate tRNA in the P-site to frameshift. (C) As in (A), but the hypomodified cognate tRNA is accepted in the A-site, and after a normal three nucleotide translocation to the P-site, the hypo modification induces slippage into the –1 or +1 frame. For clarity, only one tRNA is depicted as residing on the ribosome, although two tRNAs are always present in the A- and P-sites or P- and E-sites.
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Fig. 2. The assay systems for measuring +1 frameshifting. (A) Measurement of A-site effect by the hypomodified tRNA. The lacZ gene is placed downstream of a short frameshifting window in such a way that β-galactosidase (b-gal) activity is a direct measurement of the frequency with which the ribosome shifts frame within the short frameshifting window. This frequency of frameshifting is dependent on the competition between the peptidyl-tRNA slipping +1 base and aa-tRNA selection at the A-site. The different test codons [NNN in (A)] are placed just downstream of the frameshifting peptidyl-tRNA. (B) Measurement of P-site effect by the hypomodified tRNA. In this case, a stop codon (most often UAG) is placed just downstream of the P-site codon and the lacZ gene is placed in the +1 frame downstream of the A-site stop codon. The P-site frameshifting competes with the release factor activity in the A-site and the β-galactosidase activity is a direct measurement of the efficiency of frameshifting.
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Fig. 3. Influence of the Q34 or mnm5s2U34 on A-site effect by hypomodified tRNA. (A) Influence of Q34 on A-site selection at two His and two Tyr codons. Although the standard error for the His codon CAU suggests a difference between wild type and mutant, the t-test analysis did not (Table I). (B) Influence of Q34 on A-site selection at CAU (His), UAU (Tyr) and AAU (Asn) codons. (C) Influence of mnm5s2U34 on A-site selection at two Lys codons. Values obtained using the mnmA mutant were normalized to those using the mnmE mutant. (D) Influence of mnm5s2U34 on two Gln codons. In (A), (C) and (D), the Pro codon CCC is present at the P-site, and in (B) the Phe codon UUU is present at the P-site. Values obtained using the mnmA mutant were normalized to those using the mnmE mutant. The modification status of tRNAs reading A-site codons is indicated above the bars. The frequency of frameshifting is expressed as the β-galactosidase activity normalized to the β-lactamase activity encoded by the bla gene present on the same plasmid as that encoding the lacZ gene.
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Fig. 4. Influence of Q34 or mnm5s2U34 tRNA modifications on P-site effect by hypomodified tRNA. (A) Influence of Q34 on slippage at two Tyr codons. (B) Influence of mnm5s2U34 on slippage at two Lys codons. (C) Influence of mnm5s2U34 on slippage at two Gln codons. The stop codon UAG was present at the A-site in all cases. The modification status of tRNA reading P-site codon is indicated above the bars. The frequency of frameshifting is expressed as a percentage of the β-galactosidase activity expressed from the test plasmid compared with that activity of a pseudowild-type β-galactosidase encoded by the control plasmid.
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Fig. 5. Influence on P-site effect of ms2io6A37-deficient tRNA. (A) Influence of ms2io6A37 on slippage at two Tyr codons. Stop codon UAG is present at the A-site. (B) As in (A), but the codon UUU is present at the P-site. (C) Influence of ms2io6A37 on slippage at Phe codon UUU. The His codon CAU is present at the A-site. The modification status of tRNA reading P-site codon is indicated above the bars.
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Fig. 6. Influence on P-site effect by m1G37-deficient tRNA. (A) Influence of m1G37 on slippage at Leu codons CUU, CUA, CUG and CUC. The value for the CUU codon is 100-fold higher than shown in the figure. (B) Influence of m1G37 on slippage at the Arg codon CGG. (C) Influence of m1G37 on slippage at the Pro codon CCG. In (A) and (B), the stop codon UAG is present in the A-site, and in (C) the stop codon UGA or UAA is present in the A-site. The modification status of tRNA reading P-site codon is indicated above the bars.
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References

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