Structural Biology of Bacterial RNA Polymerase

Murakami, Katsuhiko

doi:10.3390/biom5020848

Cited by 101 publications

(122 citation statements)

References 88 publications

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“…High-resolution X-ray crystal structures are available for the T. aquaticus and E. coli RNA polymerases (12,14,16,18,20), and this proline occurs in the highly conserved D region that forms the RNA exit channel and in close proximity to amino acids found to complex with rifampin (16). The equivalent mutations of P564 in E. coli and P519 in S. aureus strain 8325-4 have been shown to confer high-level resistance to rifampin (16,24).…”

Section: Resultsmentioning

confidence: 99%

“…Structural studies of transcription initiation have revealed new insights into promoter recognition and the steps leading to open promoter formation (18,23,87,89). Promoter recognition is thought to be mediated by sigma factor-DNA interaction and core promoter elements comprising Ϫ35, Ϫ10, and extended Ϫ10 regions (12,23); however, a high-resolution study of an open promoter complex with Thermus thermophilus holoenzyme recently revealed the presence of a new determinant called the core recognition element (CRE) that is thought to provide sequence-specific promoter recognition of the Ϫ4 to ϩ2 region of the nontemplate DNA strand (89).…”

Section: Discussionmentioning

confidence: 99%

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Rifampin Resistance rpoB Alleles or Multicopy Thioredoxin/Thioredoxin Reductase Suppresses the Lethality of Disruption of the Global Stress Regulator spx in Staphylococcus aureus

et al. 2016

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Staphylococcus aureus is capable of causing a remarkable spectrum of disease, ranging from mild skin eruptions to life-threatening infections. The survival and pathogenic potential of S. aureus depend partly on its ability to sense and respond to changes in its environment. Spx is a thiol/oxidative stress sensor that interacts with the C-terminal domain of the RNA polymerase RpoA subunit, leading to changes in gene expression that help sustain viability under various conditions. Using genetic and deep-sequencing methods, we show that spx is essential in S. aureus and that a previously reported ⌬spx strain harbored suppressor mutations that allowed it to grow without spx. One of these mutations is a single missense mutation in rpoB (a P-to-L change at position 519 encoded by rpoB [rpoB-P519L]) that conferred high-level resistance to rifampin. This mutation alone was found to be sufficient to bypass the requirement for spx. The generation of rifampin resistance libraries led to the discovery of an additional rpoB mutation, R484H, which supported strains with the spx disruption. Other rifampin resistance mutations either failed to support the ⌬spx mutant or were recovered at unexpectedly low frequencies in genetic transduction experiments. The amino acid residues encoded by rpoB-P519L and -R484H map in close spatial proximity and comprise a highly conserved region of RpoB. We also discovered that multicopy expression of either trxA (encoding thioredoxin) or trxB (encoding thioredoxin reductase) supports strains with the deletion of spx. Our results reveal intriguing properties, especially of RNA polymerase, that compensate for the loss of an essential gene that is a key mediator of diverse processes in S. aureus, including redox and thiol homeostasis, antibiotic resistance, growth, and metabolism. IMPORTANCEThe survival and pathogenicity of S. aureus depend on complex genetic programs. An objective for combating this insidious organism entails dissecting genetic regulatory circuits and discovering promising new targets for therapeutic intervention. In this study, we discovered that Spx, an RNA polymerase-interacting stress regulator implicated in many stress responses in S. aureus, including responses to oxidative and cell wall antibiotics, is essential. We describe two mechanisms that suppress the lethality of spx disruption. One mechanism highlights how only certain rifampin resistance-encoding alleles of RpoB confer new properties on RNA polymerase, with important mechanistic implications. We describe additional stress conditions where the loss of spx is deleterious, thereby highlighting Spx as a multifaceted regulator and attractive drug discovery target. Staphylococcus aureus is a major human pathogen that causes a diversity of disease ranging from relatively minor to invasive and systemic disease with significant morbidity and mortality (1-7). S. aureus is common both in the community and in hospital environments and has the ability to transiently colonize the skin and mucous membranes of most humans,...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Rifampin Resistance rpoB Alleles or Multicopy Thioredoxin/Thioredoxin Reductase Suppresses the Lethality of Disruption of the Global Stress Regulator spx in Staphylococcus aureus

et al. 2016

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“…The bacterial RNAP holoenzyme nominally consists of 6 subunits: the ␤ and ␤= subunits that form the active site, an ␣ subunit dimer, an subunit, and a reversibly-binding subunit called (Fig. 1a) (Feklístov et al 2014;Murakami 2015;Ruff et al 2015). In most bacterial species, several different factors recognize cognate promoter sequences and ensure that all functional genes in the genome are expressed when required for cellular viability.…”

Section: Introductionmentioning

confidence: 99%

The essential activities of the bacterial sigma factor

et al. 2017

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Abstract:Transcription is the first and most heavily regulated step in gene expression. Sigma () factors are general transcription factors that reversibly bind RNA polymerase (RNAP) and mediate transcription of all genes in bacteria. Factors play 3 major roles in the RNA synthesis initiation process: they (i) target RNAP holoenzyme to specific promoters, (ii) melt a region of double-stranded promoter DNA and stabilize it as a single-stranded open complex, and (iii) interact with other DNA-binding transcription factors to contribute complexity to gene expression regulation schemes. Recent structural studies have demonstrated that when factors bind promoter DNA, they capture 1 or more nucleotides that are flipped out of the helical DNA stack and this stabilizes the promoter open-complex intermediate that is required for the initiation of RNA synthesis. This review describes the structure and function of the 70 family of proteins and the essential roles they play in the transcription process.Key words: transcription, bacteria, RNA polymerase, sigma factor, gene expression.

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“…Over the past 18 years, and ongoing, impressive efforts in X-ray crystallography, transitioning to cryo-electron microscopy (cryo-EM), have provided deep insights into the structural biology of the cellular MSDDRPs, including their architecture, evolution, structure-function relationships, and dynamics. Growing numbers of such studies have covered the enzymes from bacteria (1,2), archaeal species (3)(4)(5), and eukaryotes , in various interaction states.…”

Section: Cellular Msddrpsmentioning

confidence: 99%

Multisubunit DNA-Dependent RNA Polymerases from Vaccinia Virus and Other Nucleocytoplasmic Large-DNA Viruses: Impressions from the Age of Structure

Mirzakhanyan

Gershon

2017

Microbiol Mol Biol Rev

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SUMMARY The past 17 years have been marked by a revolution in our understanding of cellular multisubunit DNA-dependent RNA polymerases (MSDDRPs) at the structural level. A parallel development over the past 15 years has been the emerging story of the giant viruses, which encode MSDDRPs. Here we link the two in an attempt to understand the specialization of multisubunit RNA polymerases in the domain of life encompassing the large nucleocytoplasmic DNA viruses (NCLDV), a superclade that includes the giant viruses and the biochemically well-characterized poxvirus vaccinia virus. The first half of this review surveys the recently determined structural biology of cellular RNA polymerases for a microbiology readership. The second half discusses a reannotation of MSDDRP subunits from NCLDV families and the apparent specialization of these enzymes by virus family and by subunit with regard to subunit or domain loss, subunit dissociability, endogenous control of polymerase arrest, and the elimination/customization of regulatory interactions that would confer higher-order cellular control. Some themes are apparent in linking subunit function to structure in the viral world: as with cellular RNA polymerases I and III and unlike cellular RNA polymerase II, the viral enzymes seem to opt for speed and processivity and seem to have eliminated domains associated with higher-order regulation. The adoption/loss of viral RNA polymerase proofreading functions may have played a part in matching intrinsic mutability to genome size.

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Structural Biology of Bacterial RNA Polymerase

Cited by 101 publications

References 88 publications

Rifampin Resistance rpoB Alleles or Multicopy Thioredoxin/Thioredoxin Reductase Suppresses the Lethality of Disruption of the Global Stress Regulator spx in Staphylococcus aureus

Rifampin Resistance rpoB Alleles or Multicopy Thioredoxin/Thioredoxin Reductase Suppresses the Lethality of Disruption of the Global Stress Regulator spx in Staphylococcus aureus

The essential activities of the bacterial sigma factor

Multisubunit DNA-Dependent RNA Polymerases from Vaccinia Virus and Other Nucleocytoplasmic Large-DNA Viruses: Impressions from the Age of Structure

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