Small subunits of RNA polymerase: localization, levels and implications for core enzyme composition

Doherty, Geoffrey P.; Fogg, Mark J.; Wilkinson, Anthony J.; Lewis, Peter J.

doi:10.1099/mic.0.041566-0

Cited by 34 publications

(39 citation statements)

References 47 publications

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“…They contain an additional subunit, d, which is encoded by the rpoE gene in the model organism Bacillus subtilis. [1] Previously, the d subunit was reported to increase the specificity of RNAP for a promoter sequence, [2,3] and, recently, we reported that the d subunit is involved in regulation of transcription initiation in response to the concentration of the initiating nucleoside triphosphate (iNTP). [4] The d subunit destabilizes the open complex, a crucial intermediate of transcription initiation.…”

Section: Introductionmentioning

confidence: 99%

Structural Study of the Partially Disordered Full‐Length δ Subunit of RNA Polymerase from Bacillus subtilis

et al. 2013

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The partially disordered δ subunit of RNA polymerase was studied by various NMR techniques. The structure of the well-folded N-terminal domain was determined based on inter-proton distances in NOESY spectra. The obtained structural model was compared to the previously determined structure of a truncated construct (lacking the C-terminal domain). Only marginal differences were identified, thus indicating that the first structural model was not significantly compromised by the absence of the C-terminal domain. Various (15) N relaxation experiments were employed to describe the flexibility of both domains. The relaxation data revealed that the C-terminal domain is more flexible, but its flexibility is not uniform. By using paramagnetic labels, transient contacts of the C-terminal tail with the N-terminal domain and with itself were identified. A propensity of the C-terminal domain to form β-type structures was obtained by chemical shift analysis. Comparison with the paramagnetic relaxation enhancement indicated a well-balanced interplay of repulsive and attractive electrostatic interactions governing the conformational behavior of the C-terminal domain. The results showed that the δ subunit consists of a well-ordered N-terminal domain and a flexible C-terminal domain that exhibits a complex hierarchy of partial ordering.

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Section: Introductionmentioning

confidence: 99%

Structural Study of the Partially Disordered Full‐Length δ Subunit of RNA Polymerase from Bacillus subtilis

et al. 2013

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“…The NTD contains four ␣-helices and an antiparallel ␤-sheet (7). Delta binds to RNAP in vivo (8), but the binding site is unknown.…”

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confidence: 99%

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

et al. 2013

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dRNA polymerase (RNAP) is an extensively studied multisubunit enzyme required for transcription of DNA into RNA, yet the ␦ subunit of RNAP remains an enigmatic protein whose physiological roles have not been fully elucidated. Here, we identify a novel, so far unrecognized function of ␦ from Bacillus subtilis. We demonstrate that ␦ affects the regulation of RNAP by the concentration of the initiating nucleoside triphosphate ([iNTP]), an important mechanism crucial for rapid changes in gene expression in response to environmental changes. Consequently, we demonstrate that ␦ is essential for cell survival when facing a competing strain in a changing environment. Hence, although ␦ is not essential per se, it is vital for the cell's ability to rapidly adapt and survive in nature. Finally, we show that two other proteins, GreA and YdeB, previously implicated to affect regulation of RNAP by [iNTP] in other organisms, do not have this function in B. subtilis. RNA polymerase (RNAP) is the key enzyme responsible for transcription of DNA into RNA. Bacterial RNAP core enzyme consists of several subunits: the ␣ dimer that holds together ␤ and ␤=, which form the catalytic center, and the subunit that binds to ␤=. This core enzyme, ␣ 2 ␤␤=, is capable of elongating but not initiating transcription. To initiate transcription, the core enzyme must associate with a subunit that allows the holoenzyme to recognize specific sequences in the DNA, i.e., promoters. Typically, several different subunits are present in the cell and direct the expression of different subsets of genes (1, 2).While the ␣ 2 ␤␤= composition is conserved across the bacterial kingdom, Gram-positive Firmicutes contain an additional subunit, ␦, which is encoded by the rpoE gene in the model bacterium Bacillus subtilis. The ␦ subunit was first reported as an endogenous protein present in RNAP from phage SP01-infected Bacillus subtilis cells, which was required for its accurate middle gene transcription (3, 4). The rpoE gene specifies a protein of 173 amino acids (aa) with a molecular mass of ϳ20.5 kDa. The protein is highly acidic (pI, 3.6) (5). As determined by circular dichroism (CD) spectroscopy, it consists of two domains: (i) the N-terminal domain (NTD), which is structured; and (ii) the C-terminal domain, which is unstructured and whose amino acid composition-stretches of glutamic and aspartic acid residues-makes it virtually a polyanion (6). The structure of the ordered N-terminal domain was recently solved based on a truncated construct consisting of the N-terminal domain containing a His tag. The NTD contains four ␣-helices and an antiparallel ␤-sheet (7). Delta binds to RNAP in vivo (8), but the binding site is unknown.The in vitro effects of ␦ on transcription were previously examined in detail in the B. subtilis system. ␦ was reported to destabilize complexes between RNAP and DNA in vitro, thus increasing RNAP's specificity for good consensus promoter sequences (9, 10). Despite this inhibitory effect, ␦ was shown to stimulate transcription on some temp...

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“…While there are highly conserved subunits of RNAP across the kingdoms, within the Firmicutes we now know that there are at least two additional small subunits of RNAP, giving a subunit composition of ␣ 2 ␤␤=␦ε in these organisms. The ␦, ε, and subunits all appear to be present at levels similar to those of the other subunits of RNAP (6,17), suggesting that they are all core subunit components.…”

Section: Discussionmentioning

confidence: 90%

, 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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Small subunits of RNA polymerase: localization, levels and implications for core enzyme composition

Cited by 34 publications

References 47 publications

Structural Study of the Partially Disordered Full‐Length δ Subunit of RNA Polymerase from Bacillus subtilis

Structural Study of the Partially Disordered Full‐Length δ Subunit of RNA Polymerase from Bacillus subtilis

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

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

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