Structure of a purine-purine wobble base pair in the decoding center of the ribosome

Murphy, F.V.; Ramakrishnan, V.

doi:10.1038/nsmb866

Cited by 146 publications

(114 citation statements)

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“…The well characterized wobble interactions with the post-transcriptionally modified nucleotide inosine that decodes C, U, and A residues at the third codon position is one such example (25). Although the third position C and U (CGC and CGU) are decoded through the purine-pyrimidine interactions I-C and I-U with tRNA ICG Arg , the third position A (CGA) is decoded through a purine-purine interaction, I-A, which results in a significantly wide base pair in the decoding helix (26). Another example is the AUA codon in E. coli, decoded by tRNA k2CAU Ile , which contains a lysidine ( k2 C) residue in its anticodon (5Ј-k2 CAU-3Ј).…”

Section: Discussionmentioning

confidence: 99%

Helix 69 Is Key for Uniformity during Substrate Selection on the Ribosome

Ortiz‐Meoz

Green

2011

Journal of Biological Chemistry

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Structural studies of ribosome complexes with bound tRNAs and release factors show considerable contacts between these factors and helix 69 (H69) of 23 S rRNA. Although biochemical and genetic studies have provided some general insights into the role of H69 in tRNA and RF selection, a detailed understanding of these contributions remains elusive. Here, we present a presteady-state kinetic analysis establishing that two distinct regions of H69 make critical contributions to substrate selection. The loop of H69 (A1913) forms contacts necessary for the efficient accommodation of a subset of natural tRNA species, whereas the base of the stem (G1922) is specifically critical for UGA codon recognition by the class 1 release factor RF2. These data define a broad and critical role for this centrally located intersubunit helix (H69) in accurate and efficient substrate recognition by the ribosome.The ribosome is the ribonucleoprotein machine responsible for the faithful translation of the genetic material into the encoded polypeptide. The core RNA components of the ribosome (the 16 S and 23 S rRNA) that reflect its earliest evolution play a key role in interactions that specify the selection of tRNAs and release factors on recognition of sense and stop codons, respectively. During translation elongation, the selection of a cognate ternary complex (composed of aa-tRNA, EFTu, and GTP) from solution is specified primarily in the small ribosomal subunit where three nucleotides in 16 S rRNA directly evaluate the geometry of the codon-anticodon interaction (1). Though different in molecular detail, the selection of class 1 release factors takes place in the same decoding center of the small ribosomal subunit where conserved protein features of the RF engage the stop codon. Thus, for both decoding events, molecular interactions in the small subunit "decoding center" are early and critical contributors to the selection of cognate substrates during the translational cycle.In addition to these interactions in the decoding center, it is believed that other molecular interactions between the ribosome and these substrates contribute to binding and specificity. Structural studies of tRNA (2-5), release factors (6 -8), and ribosome recycling factor (9, 10) bound to the ribosome reveal that H69 2 of the large subunit rRNA makes extensive contacts with all of these factors. This universally conserved helix forms an intersubunit bridge that is proximal to the substrate binding cleft and directly contacts the functionally critical h44 of 16 S rRNA which contains two of the three nucleotides known to directly interact with the decoding helix (codon-anticodon) during tRNA selection (A1492 and A1493).What is the specific nature of these molecular interactions, and what do they suggest about the role of H69 in ribosome function? H69 contacts incoming tRNA in both the pre-and postaccommodation steps near positions 25/26 of the D stem and position 38, located near the anticodon stem; tRNA mutations at these positions are linked to miscoding pheno...

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Section: Discussionmentioning

confidence: 99%

Helix 69 Is Key for Uniformity during Substrate Selection on the Ribosome

Ortiz‐Meoz

Green

2011

Journal of Biological Chemistry

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“…Likewise, the rates of GTP hydrolysis on near-cognate codons differ by as much as 20-fold; the same is true for peptide bond formation (figure 2) [30]. Also tRNA modifications play an important role by expanding the repertoire of unconventional base pairs at the third codon position beyond the wobble rules; such codon-anticodon complexes can be treated as 'almost-cognate' and are much less disfavoured than the near-cognate ones [44,47]. These variations in the rates of decoding, together with the differences in the concentrations of individual cognate and near-cognate tRNAs, result in different translation rates-and possibly misreading frequencies-on individual codons.…”

Section: Kinetic Discrimination Of Incorrect Trnasmentioning

confidence: 99%

Evolutionary optimization of speed and accuracy of decoding on the ribosome

Wohlgemuth

Pohl

Mittelstaet

et al. 2011

Phil. Trans. R. Soc. B

130

156

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Speed and accuracy of protein synthesis are fundamental parameters for the fitness of living cells, the quality control of translation, and the evolution of ribosomes. The ribosome developed complex mechanisms that allow for a uniform recognition and selection of any cognate aminoacyl-tRNA (aa-tRNA) and discrimination against any near-cognate aa-tRNA, regardless of the nature or position of the mismatch. This review describes the principles of the selection-kinetic partitioning and induced fit-and discusses the relationship between speed and accuracy of decoding, with a focus on bacterial translation. The translational machinery apparently has evolved towards high speed of translation at the cost of fidelity.Keywords: ribosome; protein synthesis; translation fidelity; tRNA; error frequency; mRNA decoding SPEED AND ACCURACY OF TRANSLATIONProtein synthesis on the ribosome is a fundamentally important process that consumes a large part of the energy resources of the cell. Ribosomes are universal macromolecular machines built of two subunits, the small subunit (30S subunit in bacteria), where mRNA decoding takes place, and the large subunit (50S subunit in bacteria), which harbours the catalytic site for peptide bond formation. The decoding and peptidyl transferase centres consist of RNA, suggesting that the ribosome originates from the RNA world. Intuitively, one would expect that the ribosome has evolved to produce proteins with maximum speed and accuracy at minimum metabolic cost. The aim of this review is to discuss the mechanisms by which this is achieved and potential limits to the optimization of the ribosome performance. Protein synthesis entails four major phases: initiation, elongation, termination and recycling. During initiation, the ribosome selects an mRNA and, assisted by initiation factors, places the initiator tRNA on the appropriate start codon in the P site. In the subsequent elongation phase, amino acids are added to the growing peptide in a cyclic process. Aminoacyl-tRNAs (aa-tRNAs) enter the ribosome in a tight complex with elongation factor Tu (EF-Tu) and guanosine-5 0 -triphosphate (GTP). Following the recognition of the codon by the anticodon of aa-tRNA and GTP hydrolysis by EF-Tu, aa-tRNA is accommodated in the A site of the 50S subunit and takes part in peptide bond formation. The rate of protein elongation in bacteria is between 4 and 22 amino acids per second at 378C [1-5]; thus, a protein of an average length of 330 amino acids [6] is completed in about 10-80 s. The times required for initiation, termination and ribosome recycling (around 1 s each [3]) are short enough to make elongation rate-limiting for protein synthesis [7]. Translation of a particular codon depends on both the nature and abundance of the respective tRNAs, particularly on the non-random use of synonymous codons and the availability of the respective isoacceptor tRNAs [8]. The overall rate of translation is limited by the codon-specific rates of cognate ternary complex delivery to the A site and is further attenuated ...

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“…We considered it possible that ribosomes might treat the CGA codon as a near-cognate mismatch for the tRNA Arg(ICG) , since the I•A interaction required to decode CGA requires an altered geometry of the anticodon to accommodate the purine-purine base pair within the decoding center of the Thermus thermophilus 30S subunits (Murphy and Ramakrishnan 2004). If so, then read-through of CGA codons might be improved by growth of yeast in the presence of the aminoglycoside paromomycin, which binds to the ribosome and is known to enhance stop codon read-through and acceptance of near-cognate tRNAs (Bonetti et al 1995;Fan-Minogue and Bedwell 2008).…”

Section: Read-through Of Cga Codons Is Improved By Treatment With Parmentioning

confidence: 99%

Translation of CGA codon repeats in yeast involves quality control components and ribosomal protein L1

Letzring

Wolf

Brule

et al. 2013

RNA

105

144

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Translation of CGA codon repeats in the yeast Saccharomyces cerevisiae is inefficient, resulting in dose-dependent reduction in expression and in production of an mRNA cleavage product, indicative of a stalled ribosome. Here, we use genetics and translation inhibitors to understand how ribosomes respond to CGA repeats. We find that CGA codon repeats result in a truncated polypeptide that is targeted for degradation by Ltn1, an E3 ubiquitin ligase involved in nonstop decay, although deletion of LTN1 does not improve expression downstream from CGA repeats. Expression downstream from CGA codons at residue 318, but not at residue 4, is improved by deletion of either ASC1 or HEL2, previously implicated in inhibition of translation by polybasic sequences. Thus, translation of CGA repeats likely causes ribosomes to stall and exploits known quality control systems. Expression downstream from CGA repeats at amino acid 4 is improved by paromomycin, an aminoglycoside that relaxes decoding specificity. Paromomycin has no effect if native tRNA Arg(ICG) is highly expressed, consistent with the idea that failure to efficiently decode CGA codons might occur in part due to rejection of the cognate tRNA Arg(ICG) . Furthermore, expression downstream from CGA repeats is improved by inactivation of RPL1B, one of two genes encoding the universally conserved ribosomal protein L1. The effects of rpl1b-Δ and of either paromomycin or tRNA Arg(ICG) on CGA decoding are additive, suggesting that the rpl1b-Δ mutant suppresses CGA inhibition by means other than increased acceptance of tRNA Arg(ICG) . Thus, inefficient decoding of CGA likely involves at least two independent defects in translation.

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Structure of a purine-purine wobble base pair in the decoding center of the ribosome

Cited by 146 publications

References 14 publications

Helix 69 Is Key for Uniformity during Substrate Selection on the Ribosome

Helix 69 Is Key for Uniformity during Substrate Selection on the Ribosome

Evolutionary optimization of speed and accuracy of decoding on the ribosome

Translation of CGA codon repeats in yeast involves quality control components and ribosomal protein L1

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