Initiation with Elongator tRNAs

Samhita, Laasya; Virumäe, Kai; Rèmme, Jaanus; Varshney, Umesh

doi:10.1128/jb.00637-13

Cited by 22 publications

(36 citation statements)

References 45 publications

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“…E. coli possesses four i-tRNA genes, three at the metZWV locus and the fourth one at the metY locus. Earlier, we showed that deletion of metZWV (encoding ∼75% of total i-tRNA) confers cold sensitivity to E. coli (23)(24)(25). Whereas the ΔmetZWV strain ( Fig.…”

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confidence: 99%

An evolutionarily conserved element in initiator tRNAs prompts ultimate steps in ribosome maturation

Shetty

Varshney

2016

Proc. Natl. Acad. Sci. U.S.A.

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Ribosome biogenesis, a complex multistep process, results in correct folding of rRNAs, incorporation of >50 ribosomal proteins, and their maturation. Deficiencies in ribosome biogenesis may result in varied faults in translation of mRNAs causing cellular toxicities and ribosomopathies in higher organisms. How cells ensure quality control in ribosome biogenesis for the fidelity of its complex function remains unclear. Using Escherichia coli, we show that initiator tRNA (i-tRNA), specifically the evolutionarily conserved three consecutive GC base pairs in its anticodon stem, play a crucial role in ribosome maturation. Deficiencies in cellular contents of i-tRNA confer cold sensitivity and result in accumulation of ribosomes with immature 3′ and 5′ ends of the 16S rRNA. Overexpression of i-tRNA in various strains rescues biogenesis defects. Participation of i-tRNA in the first round of initiation complex formation licenses the final steps of ribosome maturation by signaling RNases to trim the terminal extensions of immature 16S rRNA.cold sensitivity | ribosome biogenesis | 3GC base pairs | RNase PH/RNAse R | 16S rRNA maturation R ibosomes are large and the most complex of the molecular machines in cells, using up to 40% of the cellular energy for their biogenesis (1). Ribosome biogenesis is a multistep process involving a coordinated network of RNA-RNA, RNA-protein, and protein-protein interactions. The process begins with the transcription of rRNA, its processing, nucleoside modifications, structural rearrangements, synthesis, and interaction of ribosomal proteins (r-proteins) (2). In cells, RNA processing enzymes, modification systems, and chaperone-like factors facilitate ribosome biogenesis. Any deficiencies in this process may produce ribosomes that fail to maintain the fidelity of protein synthesis and cause cellular toxicity/ death (3). Deficiencies in biogenesis factors impart cold sensitivity to Escherichia coli and affect bacterial drug resistance and virulence. In humans, genetic defects resulting in imperfect ribosome biogenesis are the cause of various ribosomopathies (4-7). Thus, a better understanding of ribosome biogenesis is a fundamental requirement to develop antimicrobials and various therapies (8-12).Of the two ribosomal subunits in E. coli, the small subunit contains a 1,542 nucleotide long rRNA (16S rRNA) along with 21 r-proteins, whereas the large subunit contains two rRNAs of 2,904 (23S rRNA) and 120 (5S rRNA) nucleotide lengths along with 33 r-proteins. The three rRNAs are synthesized as parts of a single transcript and the assembly of r-proteins begins cotranscriptionally. As the assembly advances, the precursor rRNA is cleaved by RNase III into immature units of 16S rRNA (as 17S), 23S rRNA (as 25S), and 5S rRNA (as 9S) rRNAs. The 17S rRNA retains unprocessed extensions of 110 and 33 nucleotides on the 5′ and 3′ ends, respectively. Trimming of the extensions by RNase G and other RNases matures 16S rRNA. Incorporation of r-proteins occurs in three stages. The primary r-proteins interact di...

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confidence: 99%

An evolutionarily conserved element in initiator tRNAs prompts ultimate steps in ribosome maturation

Shetty

Varshney

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…For example, in our earlier studies, we showed that overproduction of the 3GC mutant tRNA fMet could also lead to a minor increase in initiation, an observation consistent with the role of 3GC base pairs in the direct binding of the tRNA fMet to the 30S ribosome. 42,43 It is also known that an extended SD stabilizes the 70S complex. 52,53 Consistent with this, we observed that kasugamycin treatment (which is known to allow 70S mode of initiation in leaderless mRNAs [54][55][56] ) offered a selective advantage to initiation with the 3GC mutant tRNA fMet from the CAT am1 mRNA with the extended SD sequence.…”

Section: Contribution Of Shine-dalgarno (Sd):anti-sd (Asd) Interactionmentioning

confidence: 96%

“…In the metZWV strain we observed that not only the unusual tRNA fMet of the mycoplasma and rhizobia origin (lacking either the first, the third or both the first and the third GC pairs) but also the elongator tRNAs initiated protein synthesis. 42,43 In fact, initiation by the unusual tRNA fMet sustained growth of E. coli in the absence of all four of the tRNA fMet genes. 27 Normally these unusual tRNAs fail to initiate in E. coli to any significant level.…”

Section: Contribution Of Cellular Initiator Trna Levelsmentioning

confidence: 98%

Is the cellular initiation of translation an exclusive property of the initiator tRNAs?

2015

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Translation of mRNAs is the primary function of the ribosomal machinery. Although cells allow for a certain level of translational errors/mistranslation (which may well be a strategic need), maintenance of the fidelity of translation is vital for the cellular function and fitness. The P-site bound initiator tRNA selects the start codon in an mRNA and specifies the reading frame. A direct P-site binding of the initiator tRNA is a function of its special structural features, ribosomal elements, and the initiation factors. A highly conserved feature of the 3 consecutive G:C base pairs (3 GC pairs) in the anticodon stem of the initiator tRNAs is vital in directing it to the P-site. Mutations in the 3 GC pairs diminish/abolish initiation under normal physiological conditions. Using molecular genetics approaches, we have identified conditions that allow initiation with the mutant tRNAs in Escherichia coli. During our studies, we have uncovered a novel phenomenon of in vivo initiation by elongator tRNAs. Here, we recapitulate how the cellular abundance of the initiator tRNA, and nucleoside modifications in rRNA are connected with the tRNA selection in the P-site. We then discuss our recent finding of how a conserved feature in the mRNA, the Shine-Dalgarno sequence, influences tRNA selection in the P-site.

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“…22 Interestingly, none of the trees showed clustering of tRNA i with elongator tRNA Met strongly suggesting against the possibility of them having a close common ancestry. Further, we have previously shown experimentally that the elongator tRNAs can also initiate 37,38 and among them, tRNA Thr , tRNA Pro and tRNA Glu were the most efficient in initiation activity. 38 The logo plot (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Further, we have previously shown experimentally that the elongator tRNAs can also initiate 37,38 and among them, tRNA Thr , tRNA Pro and tRNA Glu were the most efficient in initiation activity. 38 The logo plot (Fig. 4a) shows that the minihelix and the anticodon regions of tRNA i are highly conserved.…”

Section: Discussionmentioning

confidence: 99%

Evolution of initiator tRNAs and selection of methionine as the initiating amino acid

Bhattacharyya

Varshney

2016

RNA Biology

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Transfer RNAs (tRNAs) have been important in shaping biomolecular evolution. Initiator tRNAs (tRNA i ), a special class of tRNAs, carry methionine (or its derivative, formyL-methionine) to ribosomes to start an enormously energy consuming but a highly regulated process of protein synthesis. The processes of tRNA i evolution, and selection of methionine as the universal initiating amino acid remain an enigmatic problem. We constructed phylogenetic trees using the whole sequence, the acceptor-TcC arm ('minihelix'), and the anticodon-dihydrouridine arm regions of tRNA i from 158 species belonging to all 3 domains of life. All the trees distinctly assembled into 3 domains of life. Large trees, generated using data for all the tRNAs of a vast number of species, fail to reveal the major evolutionary events and identity of the probable elongator tRNA sequences that could be ancestor of tRNA i . Therefore, we constructed trees using the minihelix or the whole sequence of species specific tRNAs, and iterated our analysis on 50 eubacterial species. We identified tRNA Pro , tRNA Glu , or tRNA Thr (but surprisingly not elongator tRNA Met ) as probable ancestors of tRNA i . We then determined the factors imposing selection of methionine as the initiating amino acid. Overall frequency of occurrence of methionine, whose metabolic cost of synthesis is the highest among all amino acids, remains almost unchanged across the 3 domains of life. Our correlation analysis shows that its high metabolic cost is independent of many physicochemical properties of the side chain. Our results indicate that selection of methionine, as the initiating amino acid was possibly a consequence of the evolution of one-carbon metabolism, which plays an important role in regulating translation initiation.

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Initiation with Elongator tRNAs

Cited by 22 publications

References 45 publications

An evolutionarily conserved element in initiator tRNAs prompts ultimate steps in ribosome maturation

An evolutionarily conserved element in initiator tRNAs prompts ultimate steps in ribosome maturation

Is the cellular initiation of translation an exclusive property of the initiator tRNAs?

Evolution of initiator tRNAs and selection of methionine as the initiating amino acid

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