Role of helix 44 of 16S rRNA in the fidelity of translation initiation

Qin, Daoming; Liu, Qi; Devaraj, Aishwarya; Fredrick, Kurt

doi:10.1261/rna.031203.111

Cited by 35 publications

(41 citation statements)

References 39 publications

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“…S2), results quite similar to those reported by Milon et al (16). The somewhat slower association kinetics seen here, compared with previous work (25), stems from the use of affinity-purified 50S subunits. When the start codon was replaced with AUC, the apparent rate of 50S docking decreased by >30-fold for both fast (0.064 s ) phases, in agreement with previous reports (16,25).…”

Section: H69supporting

confidence: 91%

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Roles of helix H69 of 23S rRNA in translation initiation

Liu

Fredrick

2015

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

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Initiation of translation involves the assembly of a ribosome complex with initiator tRNA bound to the peptidyl site and paired to the start codon of the mRNA. In bacteria, this process is kinetically controlled by three initiation factors-IF1, IF2, and IF3. Here, we show that deletion of helix H69 (ΔH69) of 23S rRNA allows rapid 50S docking without concomitant IF3 release and virtually eliminates the dependence of subunit joining on start codon identity. Despite this, overall accuracy of start codon selection, based on rates of formation of elongation-competent 70S ribosomes, is largely uncompromised in the absence of H69. Thus, the fidelity function of IF3 stems primarily from its interplay with initiator tRNA rather than its anti-subunit association activity. While retaining fidelity, ΔH69 ribosomes exhibit much slower rates of overall initiation, due to the delay in IF3 release and impedance of an IF3-independent step, presumably initiator tRNA positioning. These findings clarify the roles of H69 and IF3 in the mechanism of translation initiation and explain the dominant lethal phenotype of the ΔH69 mutation.T ranslation initiation can be divided into two major stages in bacteria. The first stage involves assembly of the 30S initiation complex (30SIC). Facilitated by three initiation factors, initiator tRNA (N-formyl-methionyl-tRNA fMet , or fMet-tRNA fMet ) binds to the peptidyl (P) site of the 30S subunit and pairs with the start codon on the mRNA. During the second stage, the 50S subunit associates with the 30SIC and triggers dissociation of the initiation factors, leaving the 70S initiation complex (70SIC) with fMet-tRNA fMet in the P site, ready for elongation. Because initiation is the rate-limiting step of translation and establishes the reading frame, efficient and accurate assembly of the 70SIC is critical for cell survival.30SIC assembly can be considered a largely random-order process, although there is a preferred kinetic pathway of ligand binding. IF2 and IF3 are generally first to bind to the 30S subunit, followed by IF1 and fMet-tRNA fMet , whereas the timing of mRNA binding depends on its sequence context and cellular concentration (1). The three initiation factors reciprocally stabilize one another in the 30SIC, and their binding induces a conformational change of the subunit, including a clockwise rotation of the head domain (2). IF1 is an 8-kDa protein that binds to helix h44, the 530 loop, and the S12 region and blocks the 30S aminoacyl (A) site (3). IF2 is a multidomain ribosomedependent GTPase that makes extensive contacts with both the 30S subunit and fMet-tRNA fMet . Domains G3 and C1 of IF2 bind helix h5 and h14 of 16S rRNA, the N-terminal domain (NTD) interacts with S16 and IF1, and domain C2 recognizes the acceptor stem and fMet moiety of fMet-tRNA fMet (2, 4). These interactions contribute to functions of IF2 in increasing the on rate of fMet-tRNA fMet binding and discriminating against elongator tRNAs (5). IF3 consists of two globular domains connected by a flexible linker (6, 7)...

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Section: H69supporting

confidence: 91%

“…LS experiments were performed essentially as described (25), as detailed in SI Methods. Rates of FRET changes were determined under the same conditions as LS experiments, except that labeled components (IF3-AF555 and 30S-DY647 or tRNA-OG488) were used.…”

Section: Methodsmentioning

confidence: 99%

Roles of helix H69 of 23S rRNA in translation initiation

Liu

Fredrick

2015

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

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“…In this case, however, the presence of IF1 and IF3 was required in order for the IF2-bound non-canonical 30S IC to adopt a conformational state in which IF2 is bound with an affinity that is 2 orders of magnitude lower relative to the analogous canonical 30S IC. This is consistent with previous biochemical data demonstrating that IF3 plays a key role in start-codon recognition 25,31–37 and, together with IF1, is required for coupling the rate of subunit joining to the codon composition of the 30S IC 26,27 . Although the IF1- and IF3-bound non-canonical 30S IC exhibited a significantly decreased affinity for IF2 relative to what is observed for the analogous canonical 30S IC, the IF2•tRNA sub-complex did not exhibit significant differences in the distribution of EFRET values that were sampled.…”

Section: 30s Ic-driven Changes In the Stability And Dynamics Of Ifsupporting

confidence: 93%

“…The IFs achieve this by increasing the rate of association and decreasing the rate of dissociation of fMet-tRNA fMet relative to competing elongator aa-tRNAs 24 . Beyond their role in ensuring the accuracy of 30S IC assembly, the IFs also collaborate to couple the rate of 50S subunit joining to the composition of the 30S IC, thereby increasing the rate of subunit joining to correctly assembled 30S ICs while decreasing the rate of subunit joining to ‘pseudo’ 30S ICs carrying an unaminoacylated tRNA fMet , unformylated Met-tRNA fMet , or an elongator aa-tRNA in the P site 24 , and ‘non-canonical’ 30S ICs assembled at a near-start codon 25–27 .…”

Section: Accurate Fmet-trnafmet and Start Codon Selection Is Ensurmentioning

confidence: 99%

The emerging role of rectified thermal fluctuations in initiator aa-tRNA- and start codon selection during translation initiation

Caban

Gonzalez

2015

Biochimie

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Decades of genetic, biochemical, biophysical, and structural studies suggest that the conformational dynamics of the translation machinery (TM), of which the ribosome is the central component, play a fundamental role in the mechanism and regulation of translation. More recently, single-molecule fluorescence resonance energy transfer (smFRET) studies have provided a unique and powerful approach for directly monitoring the real-time dynamics of the TM. Indeed, smFRET studies of the elongation stage of translation have significantly enriched our understanding of the mechanisms through which stochastic, thermally driven conformational fluctuations of the TM are exploited to drive and regulate the individual steps of translation elongation1. Beyond translation elongation, smFRET studies of the conformational dynamics of the initiation stage of translation offer great potential for providing mechanistic information that has thus far remained difficult or impossible to obtain using traditional methods. This is particularly true of the mechanisms through which the accuracy of initiator tRNA- and start codon selection is established during translation initiation. Given that translation initiation is a major checkpoint for regulating the translation of mRNAs, obtaining such mechanistic information holds great promise for our understanding of the translational regulation of gene expression. Here, we provide an overview of the bacterial translation initiation pathway, summarize what is known regarding the biochemical functions of the IFs, and discuss various new and exciting mechanistic insights that have emerged from several recently published smFRET studies of the mechanisms that guide initiator tRNA- and start codon selection during translation initiation. These studies provide a springboard for future investigations of the conformational dynamics of the more complex eukaryotic translation initiation pathway and mechanistic studies of the role of translational regulation of gene expression in human health and disease.

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“…This raises the question as to how well the resistance methyltransferases modifying G1405 and A1408 can be accommodated in this functionally crowded region of the ribosome. Mutations at the decoding site, in particular, at G1405 and A1408 (O'Connor et al 1997), and lack of endogenous methylation (Roy-Chaudhuri et al 2010;Qin et al 2012) can alter translation fidelity. However, the impact of exogenous methylation at these positions has not been studied.…”

Section: Introductionmentioning

confidence: 99%

Aminoglycoside resistance 16S rRNA methyltransferases block endogenous methylation, affect translation efficiency and fitness of the host

Lioy

Goussard

Guérineau

et al. 2014

RNA

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In Gram-negative bacteria, acquired 16S rRNA methyltransferases ArmA and NpmA confer high-level resistance to all clinically useful aminoglycosides by modifying, respectively, G1405 and A1408 in the A-site. These enzymes must coexist with several endogenous methyltransferases that are essential for fine-tuning of the decoding center, such as RsmH and RsmI in Escherichia coli, which methylate C1402 and RsmF C1407. The resistance methyltransferases have a contrasting distribution-ArmA has spread worldwide, whereas a single clinical isolate producing NpmA has been reported. The rate of dissemination of resistance depends on the fitness cost associated with its expression. We have compared ArmA and NpmA in isogenic Escherichia coli harboring the corresponding structural genes and their inactive point mutants cloned under the control of their native constitutive promoter in the stable plasmid pGB2. Growth rate determination and competition experiments showed that ArmA had a fitness cost due to methylation of G1405, whereas NpmA conferred only a slight disadvantage to the host due to production of the enzyme. MALDI MS indicated that ArmA impeded one of the methylations at C1402 by RsmI, and not at C1407 as previously proposed, whereas NpmA blocked the activity of RsmF at C1407. A dual luciferase assay showed that methylation at G1405 and A1408 and lack of methylation at C1407 affect translation accuracy. These results indicate that resistance methyltransferases impair endogenous methylation with different consequences on cell fitness.

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Role of helix 44 of 16S rRNA in the fidelity of translation initiation

Cited by 35 publications

References 39 publications

Roles of helix H69 of 23S rRNA in translation initiation

Roles of helix H69 of 23S rRNA in translation initiation

The emerging role of rectified thermal fluctuations in initiator aa-tRNA- and start codon selection during translation initiation

Aminoglycoside resistance 16S rRNA methyltransferases block endogenous methylation, affect translation efficiency and fitness of the host

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