Novel mechanism of gene regulation: the protein Rv1222 of<i>Mycobacterium tuberculosis</i>inhibits transcription by anchoring the RNA polymerase onto DNA

Rudra, Paulami; Prajapati, Ranjit Kumar; Banerjee, Rajdeep; Sengupta, Shreya; Mukhopadhyay, Jayanta

doi:10.1093/nar/gkv516

Cited by 11 publications

(19 citation statements)

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“…Finally, protein Rv1222 of Mycobacterium tuberculosis that interacts with both RNAP and DNA inhibits transcription by anchoring the RNAP onto DNA (Rudra et al. 2015). The positively charged C-terminal tail of the Rv1222 interacts with DNA and slows down the RNAP movement during transcription elongation.…”

Section: Introductionmentioning

confidence: 99%

Diversity, versatility and complexity of bacterial gene regulation mechanisms: opportunities and drawbacks for applications in synthetic biology

Bervoets

Charlier

2019

FEMS Microbiology Reviews

132

101

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Gene expression occurs in two essential steps: transcription and translation. In bacteria, the two processes are tightly coupled in time and space, and highly regulated. Tight regulation of gene expression is crucial. It limits wasteful consumption of resources and energy, prevents accumulation of potentially growth inhibiting reaction intermediates, and sustains the fitness and potential virulence of the organism in a fluctuating, competitive and frequently stressful environment. Since the onset of studies on regulation of enzyme synthesis, numerous distinct regulatory mechanisms modulating transcription and/or translation have been discovered. Mostly, various regulatory mechanisms operating at different levels in the flow of genetic information are used in combination to control and modulate the expression of a single gene or operon. Here, we provide an extensive overview of the very diverse and versatile bacterial gene regulatory mechanisms with major emphasis on their combined occurrence, intricate intertwinement and versatility. Furthermore, we discuss the potential of well-characterized basal expression and regulatory elements in synthetic biology applications, where they may ensure orthogonal, predictable and tunable expression of (heterologous) target genes and pathways, aiming at a minimal burden for the host.

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Section: Introductionmentioning

confidence: 99%

Diversity, versatility and complexity of bacterial gene regulation mechanisms: opportunities and drawbacks for applications in synthetic biology

Bervoets

Charlier

2019

FEMS Microbiology Reviews

132

101

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“…It is possible that AbmR interacts directly with RNAP or Rho to affect termination, or recruits an as yet undefined trans‐acting factor that modulates Mcr11 termination in Mtb. For example, Rv1222 is a mycobacterial DNA‐binding protein that was found to slow the rate of RNA synthesis through direct interaction with RNAP, and AbmR may function in a similar manner to promote termination at the mcr11 locus (Rudra, Prajapati, Banerjee, Sengupta, & Mukhopadhyay, ).…”

Section: Discussionmentioning

confidence: 99%

Small RNA Mcr11 requires the transcription factor AbmR for stable expression and regulates genes involved in the central metabolism of Mycobacterium tuberculosis

Girardin

McDonough

2019

Molecular Microbiology

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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 chronic infections in mice. We found that stable expression of Mcr11 requires multiple factors specific to TB‐complex bacteria, including the AbmR transcription factor. Bioinformatic analyses used to predict regulatory targets of Mcr11 identified 7–11 nucleotide regions with potential for direct base‐pairing with Mcr11 immediately upstream of Rv3282, fadA3, and lipB. mcr11‐dependent regulation of these genes was demonstrated using qRT‐PCR and found to be responsive to the presence of fatty acids. Mutation of the putative Mcr11 base‐pairing site upstream of lipB in a promoter reporter strain resulted in significant de‐repression of lipB expression, similar to that observed in mcr11‐deleted Mtb. These studies establish Mcr11’s roles in regulating growth and central metabolism in Mtb. Our finding that multiple TB‐complex‐specific factors are required for production of stable Mcr11 also emphasizes the need to better understand mechanisms of sRNA expression and stability in TB.

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“…FeBABE-mediated Protein-DNA Footprinting Assay-The single-cysteine (at position 51) derivative of ␦ was reacted with FeBABE (Dojindo Molecular Technologies Inc., Japan) in 1:5 molar ratio as described by Rudra et al (17). The unused probes were removed by gel filtration P-6 column (Bio-Rad), pre-equilibrated with buffer (20 mM Tris-Cl, pH 8.0, 0.2 M NaCl).…”

Section: Fluorescence Anisotropy Assaysmentioning

confidence: 99%

“…Note that 0.1-0.2 M labeled ␦ was used in FeBABE footprinting assay, 10-fold less than the amount used in DNaseI footprinting to reduce the nonspecific binding of ␦ to DNA. The cleavage reaction and purification of the products were performed as in Rudra et al (17). The products were run on 6% urea-PAGE gel.…”

Section: Fluorescence Anisotropy Assaysmentioning

confidence: 99%

Bacillus subtilis δ Factor Functions as a Transcriptional Regulator by Facilitating the Open Complex Formation

Prajapati

Sengupta

Rudra

et al. 2016

Journal of Biological Chemistry

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Most bacterial RNA polymerases (RNAP) contain five conserved subunits, viz. 2␣, ␤, ␤, and . However, in many Grampositive bacteria, especially in fermicutes, RNAP is associated with an additional factor, called ␦. For over three decades since its identification, it had been thought that ␦ functioned as a subunit of RNAP to enhance the level of transcripts by recycling RNAP. In support of the previous observations, we also find that ␦ is involved in recycling of RNAP by releasing the RNA from the ternary complex. We further show that ␦ binds to RNA and is able to recycle RNAP when the length of the nascent RNA reaches a critical length. However, in this work we decipher a new function of ␦. Performing biochemical and mutational analysis, we show that Bacillus subtilis ␦ binds to DNA immediately upstream of the promoter element at A-rich sequences on the abrB and rrnB1 promoters and facilitates open complex formation. As a result, ␦ facilitates RNAP to initiate transcription in the second scale, compared with minute scale in the absence of ␦. Using transcription assay, we show that ␦-mediated recycling of RNAP cannot be the sole reason for the enhancement of transcript yield. Our observation that ␦ does not bind to RNAP holo enzyme but is required to bind to DNA upstream of the ؊35 promoter element for transcription activation suggests that ␦ functions as a transcriptional regulator.Transcription is the first step in gene regulation in bacteria in which RNA polymerase (RNAP) 3 together with different factors and transcriptional regulators control the gene expression. Bacterial RNAP core enzyme contains five conserved subunits: 2 ␣, ␤, ␤Ј, and . A specificity factor associates with RNAP core enzyme to form RNAP holo enzyme that is able to recognize and initiate transcription at promoters.In certain Gram-positive bacteria, including Bacillus subtilis and Staphylococcus aureus, an additional factor, called ␦, is associated with RNAP. The ␦ factor was first identified in 1975 during the purification of RNAP from phage (SP01)-infected B. subtilis (1). The protein copurified with RNAP, and therefore it was thought that ␦ functions as a subunit of RNAP. Attempts were made to characterize the functional role of the protein in transcription. Several reports suggested that ␦ was involved in promoter selection (2-5) and functioned together with A as an initiation subunit of RNAP (6, 7) or as an allosteric modulator of RNAP conformation in both initiation and the RNAP core recycling phase (5). Other reports showed that ␦ and A bind to RNAP core with negative cooperativity (8, 9), and ␦ has no effect on transcription initiation, the rate of elongation, or termination (5). Using in vitro transcription assays, several groups showed that ␦ enhances the production of transcripts from certain promoters. This increase in transcript yield in the presence of ␦ is attributed to the recycling of RNAP possibly by ␦-mediated release of RNAP from the elongation complex following transcription termination or by inhibiting the formation of s...

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Novel mechanism of gene regulation: the protein Rv1222 ofMycobacterium tuberculosisinhibits transcription by anchoring the RNA polymerase onto DNA

Cited by 11 publications

References 50 publications

Diversity, versatility and complexity of bacterial gene regulation mechanisms: opportunities and drawbacks for applications in synthetic biology

Diversity, versatility and complexity of bacterial gene regulation mechanisms: opportunities and drawbacks for applications in synthetic biology

Small RNA Mcr11 requires the transcription factor AbmR for stable expression and regulates genes involved in the central metabolism of Mycobacterium tuberculosis

Bacillus subtilis δ Factor Functions as a Transcriptional Regulator by Facilitating the Open Complex Formation

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