The δ Subunit of RNA Polymerase Is Required for Rapid Changes in Gene Expression and Competitive Fitness of the Cell

Rabatinová, Alžběta; Šanderová, Hana; Matějčková, Jitka Jirát; Korelusová, Jana; Sojka, Ludek; Barvı́k, Ivan; Papoušková, Veronika; Sklenář, Vladimı́r; Žı́dek, Lukáš; Krásný, Libor

doi:10.1128/jb.00188-13

Cited by 47 publications

(63 citation statements)

References 54 publications

(50 reference statements)

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“…1F). We note that this result contradicts the previous observation by Rabatinová et al (12). The above observations that (i) ␦ binds to RNAP core but fails to induce core-mediated transcription and (ii) ␦ does not show any affinity toward RNAP holo but requires RNAP holo for its function suggested that ␦ may function as a transcriptional regulator.…”

Section: ␦ Has No Effect On Rnap Core-mediated Transcription But Enhacontrasting

confidence: 57%

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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Section: ␦ Has No Effect On Rnap Core-mediated Transcription But Enhacontrasting

confidence: 57%

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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“…While the stronger of these two promoters was largely unaffected by RpoE, the weaker promoter showed significantly decreased expression in the presence of this factor. In order to explain the effect of RpoE on promoter selection, Rabatinova et al (8) investigated the role of initiating nucleotide triphosphate (iNTP) recognition by RNAP and RNAPRpoE complexes. iNTP is required to stabilize open complex formation and therefore to initiate transcription.…”

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confidence: 99%

The δ Subunit of RNA Polymerase Guides Promoter Selectivity and Virulence in Staphylococcus aureus

et al. 2014

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In Gram-positive bacteria, and particularly the Firmicutes, the DNA-dependent RNA polymerase (RNAP) complex contains an additional subunit, termed the ␦ factor, or RpoE. This enigmatic protein has been studied for more than 30 years for various organisms, but its function is still not well understood. In this study, we investigated its role in the major human pathogen Staphylococcus aureus. We showed conservation of important structural regions of RpoE in S. aureus and other species and demonstrated binding to core RNAP that is mediated by the ␤ and/or ␤= subunits. To identify the impact of the ␦ subunit on transcription, we performed transcriptome sequencing (RNA-seq) analysis and observed 191 differentially expressed genes in the rpoE mutant. Ontological analysis revealed, quite strikingly, that many of the downregulated genes were known virulence factors, while several mobile genetic elements (SaPI5 and prophage SA3usa) were strongly upregulated. Phenotypically, the rpoE mutant had decreased accumulation and/or activity of a number of key virulence factors, including alpha toxin, secreted proteases, and Panton-Valentine leukocidin (PVL). We further observed significantly decreased survival of the mutant in whole human blood, increased phagocytosis by human leukocytes, and impaired virulence in a murine model of infection. Collectively, our results demonstrate that the ␦ subunit of RNAP is a critical component of the S. aureus transcription machinery and plays an important role during infection. Bacterial gene transcription is a complex, multifactorial process that involves several key enzymes and regulatory elements. It is driven by the activity of DNA-dependent RNA polymerase (RNAP) and its associated proteins, which form a multisubunit enzyme consisting of one ␤ subunit, one ␤= subunit, two identical ␣ subunits, and one subunit (reviewed in reference 1). Together these form the RNAP apoenzyme, which is able to perform RNA elongation and termination; however, initiation requires the involvement of a factor. Typically, most bacterial species harbor several different factors, a primary one ( A or 70 ) that mediates housekeeping gene transcription and a variety of alternative sigma factors, which aid in the response to unfavorable environmental conditions and stress.In certain Gram-positive species and particularly the Firmicutes (see Fig. S1 in the supplemental material), an additional RNAP subunit is present, termed the ␦ factor, or RpoE (2). In Bacillus subtilis, the 173-amino-acid delta subunit has been shown to reduce nonspecific binding of RNAP to DNA and lead to an elevated preference for DNA regions that include promoter sequences (3, 4). In early experiments, it was shown that RpoE has a role specifically confined to promoter selection and influences the ability of RNAP to form open promoter complexes (5, 6). Fulllength RpoE has also been described as displacing nucleic acids from RNAP-DNA or RNAP-RNA complexes in vitro, which may explain the decreased affinity of RNAP-RpoE for nonpromoter regions. Thi...

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“…As with ␦ and , ε is not essential for viability and, despite much work, the functional role of the ␦ and subunits is only now being uncovered (2,4,49). No change in the transcriptome of B. subtilis could be detected on deletion of ε or any effect of ε in in vitro transcription assays, and additional studies are needed to fully elucidate its functional role in transcription.…”

Section: Discussionmentioning

confidence: 99%

“…In the model Gram-positive organism Bacillus subtilis, core RNAP has the subunit composition ␣ 2 ␤␤=␦ 1 2 (2). The ␦ subunit is encoded by the gene rpoE and is involved in RNAP promoter specificity and recycling (2)(3)(4). The ␦ subunit appears to be conserved across the Gram-positive bacteria, and despite a high cellular abundance and documented roles in gene expression, deletion strains do not exhibit a strong phenotype (3)(4)(5).…”

mentioning

confidence: 99%

“…The ␦ subunit is encoded by the gene rpoE and is involved in RNAP promoter specificity and recycling (2)(3)(4). The ␦ subunit appears to be conserved across the Gram-positive bacteria, and despite a high cellular abundance and documented roles in gene expression, deletion strains do not exhibit a strong phenotype (3)(4)(5). The occurrence of two subunits in Bacillus subtilis at approximately 11 kDa ( 1 ) and 9 kDa ( 2 ) has been known for some time.…”

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confidence: 99%

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, a New Subunit of RNA Polymerase Found in Gram-Positive Bacteria

Keller¹,

Yang²,

Wiedermannová³

et al. 2014

Journal of Bacteriology

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b; INRA, UMR-1319 Micalis, Jouy-en-Josas, France c RNA polymerase in bacteria is a multisubunit protein complex that is essential for gene expression. We have identified a new subunit of RNA polymerase present in the high-A؉T Firmicutes phylum of Gram-positive bacteria and have named it . Previously had been identified as a small protein ( 1 ) that copurified with RNA polymerase. We have solved the structure of by X-ray crystallography and show that it is not an subunit. Rather, bears remarkable similarity to the Gp2 family of phage proteins involved in the inhibition of host cell transcription following infection. Deletion of shows no phenotype and has no effect on the transcriptional profile of the cell. Determination of the location of within the assembly of RNA polymerase core by single-particle analysis suggests that it binds toward the downstream side of the DNA binding cleft. Due to the structural similarity of with Gp2 and the fact they bind similar regions of RNA polymerase, we hypothesize that may serve a role in protection from phage infection.T ranscription in bacteria is a tightly regulated and cyclic process carried out by the highly conserved multisubunit enzyme RNA polymerase (RNAP). The majority of work on bacterial RNAP has been performed in the Gram-negative bacterium Escherichia coli. The core E. coli RNAP complex consists of two ␣ subunits and single ␤, ␤=, and subunits (1). However, there are differences throughout the eubacteria in the subunit composition of RNAP. In the model Gram-positive organism Bacillus subtilis, core RNAP has the subunit composition ␣ 2 ␤␤=␦ 1 2 (2). The ␦ subunit is encoded by the gene rpoE and is involved in RNAP promoter specificity and recycling (2-4). The ␦ subunit appears to be conserved across the Gram-positive bacteria, and despite a high cellular abundance and documented roles in gene expression, deletion strains do not exhibit a strong phenotype (3-5). The occurrence of two subunits in Bacillus subtilis at approximately 11 kDa ( 1 ) and 9 kDa ( 2 ) has been known for some time. The 2 subunit is encoded by rpoZ (synonym, yloH) and is similar to the subunit present throughout eubacteria (6-8). The 1 subunit has been referred to as the second analogous subunit simply because it has a size similar to that of the true subunit (9-11). This annotation has been accepted despite there being no evidence to suggest that these two proteins are related in function. Using mTRAQ (mass differential tags for relative and absolute quantification) mass spectrometry, it has been shown that both of the subunits are approximately equimolar with the ␤ subunit (6). Purification of RNAP shows that both subunits remain tightly bound to RNAP even after anion-exchange chromatography (8).We have determined the structure of 1 , encoded by the ykzG gene, examined the phenotype generated on its deletion, and determined its location on RNAP. The ykzG gene is present in an operon with the physiologically important RNase RNaseJ1 (RnjA) (12), and the two proteins are transcriptionally and tra...

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The δ Subunit of RNA Polymerase Is Required for Rapid Changes in Gene Expression and Competitive Fitness of the Cell

Cited by 47 publications

References 54 publications

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

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

The δ Subunit of RNA Polymerase Guides Promoter Selectivity and Virulence in Staphylococcus aureus

, a New Subunit of RNA Polymerase Found in Gram-Positive Bacteria

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