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Hierarchical mechanism of amino acid sensing by the T-box riboswitch.

ABSTRACT: In Gram-positive bacteria, T-box riboswitches control gene expression to maintain the cellular pools of aminoacylated tRNAs essential for protein biosynthesis. Co-transcriptional binding of an uncharged tRNA to the riboswitch stabilizes an antiterminator, allowing transcription read-through, whereas an aminoacylated tRNA does not. Recent structural studies have resolved two contact points between tRNA and Stem-I in the 5' half of the T-box riboswitch, but little is known about the mechanism empowering transcriptional control by a small, distal aminoacyl modification. Using single-molecule fluorescence microscopy, we have probed the kinetic and structural underpinnings of tRNA binding to a glycyl T-box riboswitch. We observe a two-step mechanism where fast, dynamic recruitment of tRNA by Stem-I is followed by ultra-stable anchoring by the downstream antiterminator, but only without aminoacylation. Our results support a hierarchical sensing mechanism wherein dynamic global binding of the tRNA body is followed by localized readout of its aminoacylation status by snap-lock-based trapping.

SUBMITTER: Suddala KC

PROVIDER: S-EPMC5951919 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Hierarchical mechanism of amino acid sensing by the T-box riboswitch.

Suddala Krishna C KC Cabello-Villegas Javier J Michnicka Malgorzata M Marshall Collin C Nikonowicz Edward P EP Walter Nils G NG

Nature communications 20180514 1

In Gram-positive bacteria, T-box riboswitches control gene expression to maintain the cellular pools of aminoacylated tRNAs essential for protein biosynthesis. Co-transcriptional binding of an uncharged tRNA to the riboswitch stabilizes an antiterminator, allowing transcription read-through, whereas an aminoacylated tRNA does not. Recent structural studies have resolved two contact points between tRNA and Stem-I in the 5' half of the T-box riboswitch, but little is known about the mechanism empo ...[more]

PMID: 29760498

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Project description:In line with the key role of methionine in protein biosynthesis initiation and many cellular processes most microorganisms have evolved mechanisms to synthesize methionine de novo. Here we demonstrate that, in the bacterial pathogen Staphylococcus aureus, a rare combination of stringent response-controlled CodY activity, T-box riboswitch and mRNA decay mechanisms regulate the synthesis and stability of methionine biosynthesis metICFE-mdh mRNA. In contrast to other Bacillales which employ S-box riboswitches to control methionine biosynthesis, the S. aureus metICFE-mdh mRNA is preceded by a 5'-untranslated met leader RNA harboring a T-box riboswitch. Interestingly, this T-box riboswitch is revealed to specifically interact with uncharged initiator formylmethionyl-tRNA (tRNAi(fMet)) while binding of elongator tRNA(Met) proved to be weak, suggesting a putative additional function of the system in translation initiation control. met leader RNA/metICFE-mdh operon expression is under the control of the repressor CodY which binds upstream of the met leader RNA promoter. As part of the metabolic emergency circuit of the stringent response, methionine depletion activates RelA-dependent (p)ppGpp alarmone synthesis, releasing CodY from its binding site and thereby activating the met leader promoter. Our data further suggest that subsequent steps in metICFE-mdh transcription are tightly controlled by the 5' met leader-associated T-box riboswitch which mediates premature transcription termination when methionine is present. If methionine supply is limited, and hence tRNAi(fMet) becomes uncharged, full-length met leader/metICFE-mdh mRNA is transcribed which is rapidly degraded by nucleases involving RNase J2. Together, the data demonstrate that staphylococci have evolved special mechanisms to prevent the accumulation of excess methionine. We hypothesize that this strict control might reflect the limited metabolic capacities of staphylococci to reuse methionine as, other than Bacillus, staphylococci lack both the methionine salvage and polyamine synthesis pathways. Thus, methionine metabolism might represent a metabolic Achilles' heel making the pathway an interesting target for future anti-staphylococcal drug development.

Project description:T-box riboswitches are a widespread class of structured noncoding RNAs in Gram-positive bacteria that regulate the expression of amino acid-related genes. They form negative feedback loops to maintain steady supplies of aminoacyl-transfer RNAs (tRNAs) to the translating ribosomes. T-box riboswitches are located in the 5' leader regions of mRNAs that they regulate and directly bind to their cognate tRNA ligands. T-boxes further sense the aminoacylation state of the bound tRNAs and, based on this readout, regulate gene expression at the level of transcription or translation. T-box riboswitches consist of two conserved domains-a 5' Stem I domain that is involved in specific tRNA recognition and a 3' antiterminator/antisequestrator (or discriminator) domain that senses the amino acid on the 3' end of the bound tRNA. Interaction of the 3' end of an uncharged but not charged tRNA with a thermodynamically weak discriminator domain stabilizes it to promote transcription readthrough or translation initiation. Recent biochemical, biophysical, and structural studies have provided high-resolution insights into the mechanism of tRNA recognition by Stem I, several structural models of full-length T-box-tRNA complexes, mechanism of amino acid sensing by the antiterminator domain, as well as kinetic details of tRNA binding to the T-box riboswitches. In addition, translation-regulating T-box riboswitches have been recently characterized, which presented key differences from the canonical transcriptional T-boxes. Here, we review the recent developments in understanding the T-box riboswitch mechanism that have employed various complementary approaches. Further, the regulation of multiple essential genes by T-boxes makes them very attractive drug targets to combat drug resistance. The recent progress in understanding the biochemical, structural, and dynamic aspects of the T-box riboswitch mechanism will enable more precise and effective targeting with small molecules. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1167-1180, 2019.

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Hierarchical mechanism of amino acid sensing by the T-box riboswitch.

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Hierarchical mechanism of amino acid sensing by the T-box riboswitch.

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