Transfer RNA–mediated regulation of ribosome dynamics during protein synthesis

Fei, Jingyi; Richard, Alison F.; Bronson, Jonathan E.; Gonzalez, Ruben L.

doi:10.1038/nsmb.2098

Cited by 55 publications

(82 citation statements)

References 57 publications

(140 reference statements)

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“…Taken together, these observations are consistent with a model in which L1 stalk and P-site tRNA Phe dynamics are coupled to intersubunit rotation in PRE/PRE -A complexes (21). It is important to note, however, that the identity of the P-site tRNA can influence the conformational equilibria of PRE/PRE -A complexes (21,23) and that the PRE -A complexes investigated here all contained exclusively tRNA Phe at the P site. Thus, future experiments using tRNAs other than tRNA Phe will be needed to determine whether and how the coupling of P-site tRNA dynamics to intersubunit rotation observed here depends on the identity of the P-site tRNA.…”

Section: Resultssupporting

confidence: 74%

“…S4A, and Tables S3 and S4). As noted above, however, the identity of the P-site tRNA can influence the conformational equilibria of PRE/PRE -A complexes (21,23) and future experiments using tRNAs other than tRNA Phe will therefore be needed to determine whether and how tRNA-mediated modulation of the cooperativity between L1-stalk dynamics and intersubunit rotation depends on the identity of the P-site tRNA.…”

Section: Resultsmentioning

confidence: 99%

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The ribosome uses cooperative conformational changes to maximize and regulate the efficiency of translation

Ning

Fei

Gonzalez

2014

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

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One of the most challenging unanswered questions regarding the structural biology of biomolecular machines such as the two-subunit ribosome is whether and how these machines coordinate seemingly independent and random conformational fluctuations to maximize and regulate their functional efficiencies. To address this question, we have used ribosome mutagenesis or a ribosome-targeting antibiotic to predictably perturb the dynamics of intersubunit rotation, a structural rearrangement of the ribosome that is essential for the translocation and ejection of ribosome-bound tRNAs during translation. Concomitantly, we have used single-molecule fluorescence resonance energy transfer (smFRET) to characterize the effects of these perturbations on the dynamics of ribosomal L1 stalk movements and ribosome-bound tRNA reconfigurations, conformational changes that are likewise essential for the translocation and ejection of tRNAs during translation. Together with the results of complementary biochemical studies, our smFRET studies demonstrate that the ribosome uses cooperative conformational changes to maximize and regulate the efficiency with which it translocates and ejects tRNAs during translation. We propose that the ribosome employs cooperative conformational changes to efficiently populate global conformational states that are productive for translation, that translation factors exploit this cooperativity as part of their mechanisms of action, and that antibiotics exploit it to maximize the potency with which they inhibit translation. It is likely that similar cooperative conformational changes underlie the function and regulation of other biomolecular machines. During the catalytic cycle of many enzymes, multiple, spatially distant enzyme structural elements must often undergo functionally important conformational changes (1). Consistent with the view that such structural rearrangements must be rapidly organized and executed to maintain catalytic efficiency, many recent studies strongly suggest that small, monomeric protein enzymes have evolved complex networks of cooperative conformational changes that coordinate the inherently stochastic conformational fluctuations of multiple structural elements in a manner that is optimal for catalysis (2). Within the context of energy landscape theory (3), such enzymes can be thought of as having evolved dynamic energy landscapes that bias enzyme conformational sampling in such a manner to maximize the efficiency of catalysis (2). Related proposals suggest that binding of allosteric effectors to enzymes remodels such energy landscapes, altering enzyme conformational sampling as part of the mechanisms through which these effectors regulate enzymatic activity (4).Unfortunately, the conformational dynamics of large, macromolecular complexes remain much more challenging to characterize than those of small, monomeric proteins (5), making it very difficult to elucidate the role that cooperative conformational changes play in the function and regulation of biomolecular machines such as the ...

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Section: Resultssupporting

confidence: 74%

Section: Resultsmentioning

confidence: 99%

The ribosome uses cooperative conformational changes to maximize and regulate the efficiency of translation

Ning

Fei

Gonzalez

2014

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

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“…Ribosomes lacking a P site tRNA or containing an oligonucleotide analog of a P site anticodon stem-loop or a P site peptidyl-tRNA rarely, if ever, undergo transitions between GS1 and GS2, nor do they significantly populate GS2, in line with the observation that such ribosomal complexes are not substrates for the translocation reaction (Joseph and Noller 1998). Most recently, Fei et al (2011) have determined how the identity of the P site tRNA can regulate the GS1/GS2 equilibrium by investigating the dynamics of a set of PRE complexes containing a series of systematically mutated tRNAs. The results of this study demonstrate that the structural stability of the P site tRNA and the specific interactions that the P site tRNA makes with the PRE ribosome control PRE complex dynamics and regulate the translocation reaction.…”

Section: Mrna-trna Translocationsupporting

confidence: 48%

Biological mechanisms, one molecule at a time

Tinoco¹,

Gonzalez²

2011

Genes Dev.

123

104

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The last 15 years have witnessed the development of tools that allow the observation and manipulation of single molecules. The rapidly expanding application of these technologies for investigating biological systems of ever-increasing complexity is revolutionizing our ability to probe the mechanisms of biological reactions. Here, we compare the mechanistic information available from single-molecule experiments with the information typically obtained from ensemble studies and show how these two experimental approaches interface with each other. We next present a basic overview of the toolkit for observing and manipulating biology one molecule at a time. We close by presenting a case study demonstrating the impact that single-molecule approaches have had on our understanding of one of life's most fundamental biochemical reactions: the translation of a messenger RNA into its encoded protein by the ribosome.Why would anybody want to study biology one molecule at a time? For the same reason some biologists study individual animals instead of populations. As Steven Chu-who, in 1997, won the Nobel Prize in Physics for developing a method to trap and manipulate individual atoms-pointed out, on average, humans have one mammary and one testicle. We are told that all electrons are identical, so measuring one at a time will tell us nothing new. However, it is less clear that all Escherichia coli ribosomes are the same. Each ribosome, a complex machine composed of three ribosomal RNA molecules and >50 ribosomal proteins, will surely have small differences in sequence, composition, covalent modification, bound ligands, and so forth. Of course, the ribosome is also not static; it is dynamic. Its two subunits rotate relative to each other and its various structural domains undergo conformational changes as it moves directionally along its messenger RNA (mRNA) template, selecting aminoacyl-transfer RNA (aa-tRNA) substrates and catalyzing the addition of each amino acid to the polypeptide chain being synthesized. Indeed, the structural dynamics involved in this process are complicated enough that they would be very difficult, if not impossible, to follow if we were restricted to only measuring the average properties of many millions of ribosomes, all simultaneously producing proteins. By watching one ribosome at work, however, we can follow its structural rearrangements as it takes each step necessary to transform a nucleotide sequence into a protein. Of course, we will be interested in watching more than one ribosome so that we can learn the range of abilities and effectiveness present among the entire population of ribosomes. Even if all of the ribosomes are identical in structure, random thermal fluctuations will cause differences in their activities. Not all of them will follow the same path through their reaction process; there may be short cuts or detours along the way. The ability to observe single molecules allows us to ask and answer questions that were impossible, or extremely difficult, to approach before. In this revi...

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“…It is interesting that, in the structure of tRNA Phe bound to E. coli 70S ribosomes in the hybrid P/E site, the anticodon helix is also in a distorted configuration, but the direction and angle of the bend is different than in the decoding complex (Dunkle et al 2011). Since the entry of tRNAs into the E site also shows a distinct sequence specificity (Fei et al 2011), it is possible that some of the tRNA consensus has evolved to optimize this step as well. Since tRNAs bound to near-cognate codons in the P site are also subject to proofreading Green 2009, 2010), it may be that that much of the tRNA consensus has evolved to maintain translational accuracy.…”

Section: Resultsmentioning

confidence: 99%

tRNA residues evolved to promote translational accuracy

Shepotinovskaya

Uhlenbeck

2013

RNA

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The decoding properties of 22 structurally conservative base-pair and base-triple mutations in the anticodon hairpin and tertiary core of Escherichia coli tRNA Ala GGC were determined under single turnover conditions using E. coli ribosomes. While all of the mutations were able to efficiently decode the cognate GCC codon, many showed substantial misreading of near-cognate GUC or ACC codons. Although all the misreading mutations were present in the sequences of other E. coli tRNAs, they were never found among bacterial tRNA Ala GGC sequences. This suggests that the sequences of bacterial tRNA Ala GGC have evolved to avoid reading incorrect codons.

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Transfer RNA–mediated regulation of ribosome dynamics during protein synthesis

Cited by 55 publications

References 57 publications

The ribosome uses cooperative conformational changes to maximize and regulate the efficiency of translation

The ribosome uses cooperative conformational changes to maximize and regulate the efficiency of translation

Biological mechanisms, one molecule at a time

tRNA residues evolved to promote translational accuracy

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