Physiological analysis of the role of truB in Escherichia coli: a role for tRNA modification in extreme temperature resistance

Kinghorn, Seonag; O’Byrne, Conor; Booth, Ian R.; Stansfield, Ian

doi:10.1099/00221287-148-11-3511

Cited by 45 publications

(44 citation statements)

References 41 publications

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“…truB KO strains are outcompeted by WT E. coli in coculture at 37°C, indicating that TruB does contribute to bacterial fitness (10). Surprisingly this fitness disadvantage of the truB KO strain can be rescued by expressing a catalytically inactive variant of TruB (10,21). This finding suggests that the presence of the TruB protein itself is important for the cell, rather than pseudouridines that are formed by TruB.…”

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confidence: 72%

RNA modification enzyme TruB is a tRNA chaperone

Keffer-Wilkes

Veerareddygari

Kothe

2016

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

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Cellular RNAs are chemically modified by many RNA modification enzymes; however, often the functions of modifications remain unclear, such as for pseudouridine formation in the tRNA TΨC arm by the bacterial tRNA pseudouridine synthase TruB. Here we test the hypothesis that RNA modification enzymes also act as RNA chaperones. Using TruB as a model, we demonstrate that TruB folds tRNA independent of its catalytic activity, thus increasing the fraction of tRNA that can be aminoacylated. By rapid kinetic stopped-flow analysis, we identified the molecular mechanism of TruB's RNA chaperone activity: TruB binds and unfolds both misfolded and folded tRNAs thereby providing misfolded tRNAs a second chance at folding. Previously, it has been shown that a catalytically inactive TruB variant has no phenotype when expressed in an Escherichia coli truB KO strain [Gutgsell N, et al. (2000) RNA 6(12):1870-1881]. However, here we uncover that E. coli strains expressing a TruB variant impaired in tRNA binding and in in vitro tRNA folding cannot compete with WT E. coli. Consequently, the tRNA chaperone activity of TruB is critical for bacterial fitness. In conclusion, we prove the tRNA chaperone activity of the pseudouridine synthase TruB, reveal its molecular mechanism, and demonstrate its importance for cellular fitness. We discuss the likelihood that other RNA modification enzymes are also RNA chaperones.lthough there is a wealth of information on RNA structure, we are just beginning to understand the RNA folding process that is often assisted by RNA chaperones (1). In contrast to many protein chaperones, RNA chaperones are not ATPases, but instead facilitate unfolding and folding of RNA directly through their interactions with RNA. In addition, the vast majority of all RNAs, including mRNAs, are posttranscriptionally modified by a plethora of RNA modification enzymes (2). Very little is known about the interplay of RNA folding and modification, although it has been speculated that RNA modification enzymes may also act as RNA chaperones (3).Despite the abundance of RNA modifications, their cellular functions are often unclear, including their possible contributions to RNA structure and stability (4). Interestingly, very few RNA modification enzymes are essential for the cell; however, many of these enzymes are conserved. The most abundant RNA modification is the conversion of uridines to pseudouridines that are found in almost all cellular RNAs (4-6). Pseudouridine formation is catalyzed by stand-alone pseudouridine synthases in all domains of life and in addition by H/ACA small ribonucleoproteins (H/ACA sRNPs) in eukaryotes and archaea (7). Remarkably, the only essential pseudouridine synthase is the eukaryotic enzyme Cbf5, the catalytic component of H/ACA sRNPs, whereas all known stand-alone pseudouridine synthases are nonessential (8). Indeed, deletion of most stand-alone pseudouridine synthases in Escherichia coli (9, 10) or Saccharomyces cerevisiae (11) does not impact cell growth under optimal conditions. Surprisingly, ev...

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confidence: 72%

RNA modification enzyme TruB is a tRNA chaperone

Keffer-Wilkes

Veerareddygari

Kothe

2016

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

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“…While tRNA modifications have been found to play significant roles in determining decoding specificity (Gustilo et al 2008) and conformational stability of tRNA molecules (for instance, see Kinghorn et al 2002;Shigi et al 2006), functional roles for rRNA modifications have not been clearly established. The location of the majority of modifications in and around the decoding and peptidyltransferase active sites of the ribosome has been interpreted to imply an important role for modifications in ribosome function (Decatur and Fournier 2002;Chow et al 2007).…”

Section: Introductionmentioning

confidence: 99%

Structural and functional studies of the Thermus thermophilus 16S rRNA methyltransferase RsmG

Gregory¹,

DeMi̇rci̇²,

Belardinelli³

et al. 2009

RNA

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The RsmG methyltransferase is responsible for N 7 methylation of G527 of 16S rRNA in bacteria. Here, we report the identification of the Thermus thermophilus rsmG gene, the isolation of rsmG mutants, and the solution of RsmG X-ray crystal structures at up to 1.5 Å resolution. Like their counterparts in other species, T. thermophilus rsmG mutants are weakly resistant to the aminoglycoside antibiotic streptomycin. Growth competition experiments indicate a physiological cost to loss of RsmG activity, consistent with the conservation of the modification site in the decoding region of the ribosome. In contrast to Escherichia coli RsmG, which has been reported to recognize only intact 30S subunits, T. thermophilus RsmG shows no in vitro methylation activity against native 30S subunits, only low activity with 30S subunits at low magnesium concentration, and maximum activity with deproteinized 16S rRNA. Cofactor-bound crystal structures of RsmG reveal a positively charged surface area remote from the active site that binds an adenosine monophosphate molecule. We conclude that an early assembly intermediate is the most likely candidate for the biological substrate of RsmG.

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“…First, the low rate constant of pseudouridine formation might be due to the absence of evolutionary pressure to further increase the rate of pseudouridine formation. While pseudouridines are the most common RNA modifications and supposed to enhance RNA structure and function, many pseudouridines and, in turn, many pseudouridine synthases, are not essential for the cell (Raychaudhuri et al 1999;Gutgsell et al 2000;Del Campo et al 2001;Kinghorn et al 2002). Second, it might even be envisioned that pseudouridine synthases have been selected to be slow.…”

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confidence: 99%

Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis

Wright

Keffer-Wilkes²,

Dobing³

et al. 2011

RNA

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Pseudouridine synthases catalyze formation of the most abundant modification of functional RNAs by site-specifically isomerizing uridines to pseudouridines. While the structure and substrate specificity of these enzymes have been studied in detail, the kinetic and the catalytic mechanism of pseudouridine synthases remain unknown. Here, the first pre-steady-state kinetic analysis of three Escherichia coli pseudouridine synthases is presented. A novel stopped-flow absorbance assay revealed that substrate tRNA binding by TruB takes place in two steps with an overall rate of 6 sec À1 . In order to observe catalysis of pseudouridine formation directly, the traditional tritium release assay was adapted for the quench-flow technique, allowing, for the first time, observation of a single round of pseudouridine formation. Thereby, the single-round rate constant of pseudouridylation (k C ) by TruB was determined to be 0.5 sec À1 . This rate constant is similar to the k cat obtained under multiple-turnover conditions in steady-state experiments, indicating that catalysis is the rate-limiting step for TruB. In order to investigate if pseudouridine synthases are characterized by slow catalysis in general, the rapid kinetic quench-flow analysis was also performed with two other E. coli enzymes, RluA and TruA, which displayed rate constants of pseudouridine formation of 0.7 and 0.35 sec À1 , respectively. Hence, uniformly slow catalysis might be a general feature of pseudouridine synthases that share a conserved catalytic domain and supposedly use the same catalytic mechanism.

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Physiological analysis of the role of truB in Escherichia coli: a role for tRNA modification in extreme temperature resistance

Cited by 45 publications

References 41 publications

RNA modification enzyme TruB is a tRNA chaperone

RNA modification enzyme TruB is a tRNA chaperone

Structural and functional studies of the Thermus thermophilus 16S rRNA methyltransferase RsmG

Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis

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