PubMed · 15578054
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Luisa Cochella, Rachel Green. 2004. Wobble during decoding: more than third-position promiscuity.. https://doi.org/10.1038/nsmb1204-1160
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Bacterial tmRNA rescues ribosomes that stall because of defective mRNAs via the trans-translation process. Although entry of the charged transfer messenger RNA (tmRNA) into the ribosome proceeded in the absence of elongation factor (EF-Tu) and in the presence of EF-Tu and the antibiotic kirromycin, evidence was found for the involvement of EF-Tu in trans-translation initiation. The polyalanine synthesis system attained by using a tmRNA variant consisting of only the tRNA-like domain revealed that it was completely dependent on the presence of SmpB and greatly enhanced by EF-Tu and EF-G. Actually, ribosome-dependent GTPase activity of EF-Tu was stimulated by the addition of SmpB and tmRNA but independently of template mRNA, demonstrating that SmpB compensates for the lack of codon-anticodon interaction during the first step of the trans-translation initiation. Based on these results, we suggest that SmpB structurally mimics the anticodon arm of tRNA and elicits GTP hydrolysis of EF-Tu upon tmRNA accommodation in the A site of the ribosome.
Life depends on the faithful translation of the genetic information into proteins. Ribosomes have developed remarkable mechanisms to ensure the accurate synthesis of proteins. In the first part of this review various types of ribosomal errors and their importance for cell-life are surveyed, while in the second part two important aspects of the ribosomal E-site for the accuracy of translation are considered: (i) The fact that usually misincorporations are not harmful for the cell, since only one in about 400 misincorporations will affect the structure and/or function of a protein, is a function of the E-site. (ii) In contrast, an extremely harmful translational error is a loss of the reading frame, resulting in an immediate loss of the genetic information. Maintenance of the reading frame is one of the most remarkable achievements of the ribosome; only once in about 30,000 elongation cycles is the reading frame lost. A cognate tRNA at the E-site is an essential prerequisite for this high precision.
The biological role of many nonessential tRNA modifications outside of the anticodon remains elusive despite their evolutionary conservation. We show here that m7G46 methyltransferase Trm8p/Trm82p acts as a hub of synthetic interactions with several tRNA modification enzymes, resulting in temperature-sensitive growth. Analysis of three double mutants indicates reduced levels of tRNA(Val(AAC)), consistent with a role of the corresponding modifications in maintenance of tRNA levels. Detailed examination of a trm8-delta trm4-delta double mutant demonstrates rapid degradation of preexisting tRNA(Val(AAC)) accompanied by its de-aminoacylation. Multiple copies of tRNA(Val(AAC)) suppress the trm8-delta trm4-delta growth defect, directly implicating this tRNA in the phenotype. These results define a rapid tRNA degradation (RTD) pathway that is independent of the TRF4/RRP6-dependent nuclear surveillance pathway. The degradation of an endogenous tRNA species at a rate typical of mRNA decay demonstrates a critical role of nonessential modifications for tRNA stability and cell survival.