Processing of the seven valine tRNAs in Escherichia coli involves novel features of RNase P

Agrawal, Ankit; Mohanty, Bijoy K.; Kushner, Sidney R.

doi:10.1093/nar/gku758

Cited by 28 publications

(51 citation statements)

References 46 publications

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“…In vitro structure−function studies demonstrate that sequence variation at 5′ leader nucleotides involved in both RNA−RNA and RNA−protein interactions can have significant functional effects on RNase P processing rates as well as cleavage site specificity. 9,21,29 For example, analysis of 5′ leader point mutations and amino acid substitutions in P protein support favorable hydrogen bonding interactions between an adenosine at N(−4) and the protein subunit of Bacillus subtilis RNase P; however, the sequence preference of E. coli RNase P at this position is diminished. 9 Additionally, mutation of the 5′ leader at N(−1) showed altered processing rates and that are partly rescued by compensatory mutations P RNA in J5/ 15.…”

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confidence: 99%

Determination of the Specificity Landscape for Ribonuclease P Processing of Precursor tRNA 5′ Leader Sequences

et al. 2016

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Maturation of tRNA depends on a single endonuclease, ribonuclease P (RNase P), to remove highly variable 5' leader sequences from precursor tRNA transcripts. Here, we use high-throughput enzymology to report multiple-turnover and single-turnover kinetics for Escherichia coli RNase P processing of all possible 5' leader sequences, including nucleotides contacting both the RNA and protein subunits of RNase P. The results reveal that the identity of N(-2) and N(-3) relative to the cleavage site at N(1) primarily control alternative substrate selection and act at the level of association not the cleavage step. As a consequence, the specificity for N(-1), which contacts the active site and contributes to catalysis, is suppressed. This study demonstrates high-throughput RNA enzymology as a means to globally determine RNA specificity landscapes and reveals the mechanism of substrate discrimination by a widespread and essential RNA-processing enzyme.

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confidence: 99%

Determination of the Specificity Landscape for Ribonuclease P Processing of Precursor tRNA 5′ Leader Sequences

et al. 2016

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“…Processing mechanisms for tRNAs and sRNAs have been defined, particularly for transcripts terminated by RITs in E. coli (57, 66-70). Recent work has demonstrated that Rho terminated transcripts have processed 3’ ends immediately downstream of a stable stem-loop (71).…”

Section: Discussionmentioning

confidence: 99%

Small RNA Mcr11 regulates genes involved in the central metabolism ofMycobacterium tuberculosisand requires 3’ sequence along with the transcription factor AbmR for stable expression

2019

Preprint

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Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis, must adapt to host-associated environments during infection by modulating gene expression. Small regulatory RNAs (sRNAs) are key regulators of bacterial gene expression, but their roles in Mtb are not well understood. Here, we address the expression and function of the Mtb sRNA Mcr11, which is associated with slow bacterial growth and latent infections in mice. We found, by using biochemical and genetic approaches, that the AbmR transcription factor and an extended region of native sequence 3’ to the mcr11 gene enhance production of mature Mcr11. Additionally, we found that expression of Mcr11 was unstable in the saprophyte Mycobacterium smegmatis, which lacks an mcr11 orthologue. Bioinformatic analyses used to predict regulatory targets of Mcr11 identified 9-11 nucleotide regions immediately upstream of Rv3282 and lipB with potential for direct base-pairing with Mcr11. mcr11-dependent regulation of Rv3282, lipB, Rv2216 and pknA was demonstrated using qRT-PCR in wild type versus mcr11-deleted Mtb and found to be responsive to the presence of fatty acids. These studies establish that Mcr11 has roles in regulating growth and central metabolism in Mtb that warrant further investigation. In addition, our finding that multiple factors are required for production of stable, mature Mcr11 emphasizes the need to study mechanisms of sRNA expression and stability in TB complex mycobacteria to understand their roles in TB pathogenesis.Author SummaryBacterial pathogens must continuously modulate their gene expression in response to changing conditions to successfully infect and survive within their hosts. Transcription factors are well known regulators of gene expression, but there is growing recognition that small RNAs (sRNAs) also have critically important roles in bacterial gene regulation. Many sRNAs have been identified in M. tuberculosis (Mtb), but little is known about their expression, regulatory targets or roles in Mtb biology. In this study, we found that the Mtb sRNA Mcr11, which is expressed at high levels in slowly replicating Mtb and during mouse infection, regulates expression of several target genes involved in central metabolism. Importantly, we also discovered that mcr11 has unexpected requirements for stable expression in mycobacteria. In particular, we identified RNA sequence elements immediately downstream of mcr11 that enhance transcription termination and production of mature Mcr11 RNA in TB-complex mycobacteria. Meanwhile, ectopic expression of Mcr11 was unstable in a non-pathogenic strain of mycobacteria, suggesting that factors specific to pathogenic mycobacteria are required for the stable production of Mcr11. These studies identify sRNA stability as a new frontier for understanding gene expression in Mtb.

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“…3C). Some require the initial removal of the Rho-independent transcription terminator by RNase E before RNase P can process the rest of the transcript (64, 65, 67). The RNase P cleavages occur at the mature 5′ termini (65), while RNase E cleavages, with the exception of the three proline tRNAs (63), leave extra nucleotides downstream of the CCA determinant (11, 66).…”

Section: Enzymes Involved In Transfer Rna Processingmentioning

confidence: 99%

“…The RNase P cleavages occur at the mature 5′ termini (65), while RNase E cleavages, with the exception of the three proline tRNAs (63), leave extra nucleotides downstream of the CCA determinant (11, 66). A surprising observation from the RNase P processed transcripts was that the enzyme cleaves the polycistronic transcripts starting from the 3′ terminus and not the 5′ terminus (64). …”

Section: Enzymes Involved In Transfer Rna Processingmentioning

confidence: 99%

“…Specifically, unlike other ribonucleases, it specifically stops at the terminal CCA because of its inhibition by the presence of C ribonucleotides within its catalytic site (69). Thus, tRNAs containing C residues downstream of CCA are more dependent on RNase PH, which is not inhibited by C residues (64). Inactivation of both RNase T and RNase PH leads to rapid accumulation of 3′ immature tRNAs which becomes substrates for PAP I (68).…”

Section: Enzymes Involved In Transfer Rna Processingmentioning

confidence: 99%

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Enzymes Involved in Posttranscriptional RNA Metabolism in Gram-Negative Bacteria

Mohanty¹,

Kushner

2018

Microbiol Spectr

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Gene expression in Gram-negative bacteria is regulated at many levels, including transcription initiation, RNA processing, RNA/RNA interactions, mRNA decay, and translational controls involving enzymes that alter translational efficiency. In this chapter we discuss the various enzymes that control transcription, translation and RNA stability through RNA processing and degradation. RNA processing is essential to generate functional RNAs, while degradation helps control the steady-state level of each individual transcript. For example, all the pre-tRNAs are transcribed with extra nucleotides at both their 5′ and 3′ termini, which are subsequently processed to produce mature tRNAs that can be aminoacylated. Similarly, rRNAs that are transcribed as part of a 30S polycistronic transcript, are matured to individual 16S, 23S and 5S rRNAs. Decay of mRNAs plays a key role in gene regulation through controlling the steady-state level of each transcript, which is essential for maintaining appropriate protein levels. In addition, degradation of both translated and non-translated RNAs recycles nucleotides to facilitate new RNA synthesis. To carry out all these reactions Gram-negative bacteria employ a large number of endonucleases, exonucleases, RNA helicases, and poly(A) polymerase as well as proteins that regulate the catalytic activity of particular ribonucleases. Under certain stress conditions an additional group of specialized endonucleases facilitate the cell’s ability to adapt and survive. Many of the enzymes, such as RNase E, RNase III, polynucleotide phosphorylase, RNase R, and poly(A) polymerase I participate in multiple RNA processing and decay pathways.

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Processing of the seven valine tRNAs in Escherichia coli involves novel features of RNase P

Cited by 28 publications

References 46 publications

Determination of the Specificity Landscape for Ribonuclease P Processing of Precursor tRNA 5′ Leader Sequences

Determination of the Specificity Landscape for Ribonuclease P Processing of Precursor tRNA 5′ Leader Sequences

Small RNA Mcr11 regulates genes involved in the central metabolism ofMycobacterium tuberculosisand requires 3’ sequence along with the transcription factor AbmR for stable expression

Enzymes Involved in Posttranscriptional RNA Metabolism in Gram-Negative Bacteria

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