Role of 16S ribosomal RNA methylations in translation initiation in Escherichia coli

Das, Gautam; Thotala, Dinesh; Kapoor, Suman; Karunanithi, Sellam; Thakur, Suman; Singh, N. S.; Varshney, Umesh

doi:10.1038/emboj.2008.20

Cited by 63 publications

(95 citation statements)

References 45 publications

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“…Earlier studies in yeast showed that although overproduction of initiator tRNA could sustain a strain lacking all five elongator tRNA Met genes, a strain lacking all its chromosomal copies of the initiator tRNA Met genes could neither be sustained by overproduction of the elongator tRNA Met nor by overproduction of initiator tRNA Met lacking the 3G-C pairs (18). The only known exceptions to the presence of the 3G-C pairs are some species of Mycoplasma (which has the smallest bacterial genomes and is most often parasitic) (11) and Rhizobium (which lives in symbiotic association).…”

Section: Discussionmentioning

confidence: 99%

“…This has led to the belief that the intact 3G-C pairs of initiator tRNA are an essential feature of living systems. The only known exceptions are in some species of Mycoplasma and Rhizobium, where the single initiator tRNA gene produces tRNAs having the first and/or last G-C pair represented by A-U and G-U, respectively (11). Understanding how initiation occurs in these species has therefore been a major puzzle in the field of translation.…”

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confidence: 99%

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Unconventional initiator tRNAs sustain Escherichia coli

Samhita

Shetty

Varshney

2012

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

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Of all tRNAs, initiator tRNA is unique in its ability to start protein synthesis by directly binding the ribosomal P-site. This ability is believed to derive from the almost universal presence of three consecutive G-C base (3G-C) pairs in the anticodon stem of initiator tRNA. Consistent with the hypothesis, a plasmid-borne initiator tRNA with one, two, or all 3G-C pairs mutated displays negligible initiation activity when tested in a WT Escherichia coli cell. Given this, the occurrence of unconventional initiator tRNAs lacking the 3G-C pairs, as in some species of Mycoplasma and Rhizobium , is puzzling. We resolve the puzzle by showing that the poor activity of unconventional initiator tRNAs in E. coli is because of competition from a large pool of the endogenous WT initiator tRNA (possessing the 3G-C pairs). We show that E. coli can be sustained on an initiator tRNA lacking the first and third G-C pairs; thereby reducing the 3G-C rule to a mere middle G-C requirement. Two general inferences following from our findings, that the activity of a mutant gene product may depend on its abundance in the cell relative to that of the WT, and that promiscuous initiation with elongator tRNAs has the potential to enhance phenotypic diversity without affecting genomic integrity, have been discussed.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Unconventional initiator tRNAs sustain Escherichia coli

Samhita

Shetty

Varshney

2012

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

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“…Furthermore, m 6 2 A1518 and m 6 2 A1519 impact initiation 67 possibly via m 3 U1498 whose backbone interacts with m 6 2 A1519, while its modified base contacts the mRNA backbone. The methyl groups of m 3 U1498 and m 4 Cm1402 form part of the binding site for the initiation codon and modulate translation initiation 13,14 by steric encumbrance and/or by preventing direct hydrogen bonds with the mRNA backbone. b, Constitutive rRNA modifications in the E. coli 70S ribosome (list adapted from ref.…”

Section: Extended Datamentioning

confidence: 99%

“…Clusters of several rRNA modifications are essential for ribosome function, whereas individual rRNA modifications have an important role in fine-tuning the active centres of the ribosome, as well as in antibiotic resistance and sensitivity 11 . Six individual rRNA modifications at the decoding centre modulate the efficiency and accuracy of translation initiation by stabilizing interactions in the P site [12][13][14] . The present structure shows that the methyl group of m 2 G966 in 16S rRNA may act as a 'backstop' for the initiator tRNA, thereby stabilizing base-pairing with the initiation codon ( Fig.…”

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Structure of the E. coli ribosome–EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM

Fischer

Neumann

Konevega

et al. 2015

Nature

362

443

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Single particle electron cryomicroscopy (cryo-EM) has recently made significant progress in high-resolution structure determination of macromolecular complexes due to improvements in electron microscopic instrumentation and computational image analysis. However, cryo-EM structures can be highly non-uniform in local resolution 1,2 and all structures available to date have been limited to resolutions above 3 Å 3,4 . Here we present the cryo-EM structure of the 70S ribosome from Escherichia coli in complex with elongation factor Tu, aminoacyl-tRNA and the antibiotic kirromycin at 2.65-2.9 Å resolution using spherical aberration (C s )-corrected cryo-EM. Overall, the cryo-EM reconstruction at 2.9 Å resolution is comparable to the best-resolved X-ray structure of the E. coli 70S ribosome 5 (2.8 Å ), but provides more detailed information (2.65 Å ) at the functionally important ribosomal core. The cryo-EM map elucidates for the first time the structure of all 35 rRNA modifications in the bacterial ribosome, explaining their roles in fine-tuning ribosome structure and function and modulating the action of antibiotics. We also obtained atomic models for flexible parts of the ribosome such as ribosomal proteins L9 and L31. The refined cryo-EM-based model presents the currently most complete high-resolution structure of the E. coli ribosome, which demonstrates the power of cryo-EM in structure determination of large and dynamic macromolecular complexes.Determining the structure of large, dynamic biological macromolecules at a uniformly high resolution provides a challenge both for X-ray crystallography and cryo-EM. Here we have used aberration-corrected cryo-EM in combination with extensive computational sorting to solve *These authors contributed equally to this work.

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“…49 Methionine is important for quorumsensing. 48,51-52 The intracellular concentrations of methionine and N 10 -formyl THF (products of one-carbon metabolism), shown to be important in protein synthesis, 53 might have acted as the indicators for energy status of the cell or the richness of the surrounding environment in the prebiotic soup. Evolutionary importance of changes in the tRNA gene and energy efficiency with bacterial growth has already been well characterized.…”

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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Role of 16S ribosomal RNA methylations in translation initiation in Escherichia coli

Cited by 63 publications

References 45 publications

Unconventional initiator tRNAs sustain Escherichia coli

Unconventional initiator tRNAs sustain Escherichia coli

Structure of the E. coli ribosome–EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM

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

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