Mutations in the CRE pocket of bacterial RNA polymerase affect multiple steps of transcription

Petushkov, Ivan; Pupov, Danil; Bass, I. A.; Kulbachinskiy, Andrey

doi:10.1093/nar/gkv504

Cited by 28 publications

(29 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3A), comparable to the effects of substitutions of key NT bases and RNAP residues that interact with these bases (11,(14)(15)(16). Consistent with the deduction that T7A1 and rrnB P1 open complexes resemble λP R I 3 and I 2 (19), the GL deletion had smaller (4.4-and 1.6-fold) effects at these promoters (Fig.…”

Section: Resultssupporting

confidence: 81%

“…The discriminator region between the −10 hexamer and the transcription start site (TSS; +1) has recently emerged as an important modulator of the open complex properties (11)(12)(13)(14)(15)(16)(17)(18). In a stable open complex formed by Thermus thermophilus RNAP, the −6 and −5 bases of the discriminator NT strand (NT DISC ) make crucial contacts with σ1.2, and βArg371 (E. coli numbering) interacts with the adjacent −4 and −3 bases (14).…”

Section: Resultsmentioning

confidence: 99%

“…Substitutions of the discriminator bases and the σ and β residues that interact with the NT strand lead to decreases in open complex stability (11,14,16). To test whether the loss of the GL-NT DISC contacts observed in the RPo structure (Fig.…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes

NandyMazumdar

Nedialkov

Svetlov

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β′ clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.D uring each round of transcription, RNA polymerase (RNAP) establishes, maintains, and finally releases contacts with the DNA template and the RNA. These interactions mediate highly selective initiation, processive elongation, and precise termination and can be tuned to enable intricate regulation during the transcription cycle. RNAP resembles a crab claw in which the two pincers composed of the β′ and β subunits form an active site cleft that accommodates the nucleic acid chains (Fig. 1). The mobile β′ clamp domain that forms one of the pincers stands out as a central regulatory feature (1,2). The open clamp likely allows the loading of the promoter DNA during initiation and the release of the template and the nascent RNA during termination. The clamp is thought to close to form an active initiation complex and to remain closed during elongation but may open partially at a hairpindependent pause site (3).The clamp movements may be linked to those of the β lobe domain, which forms a part of the second pincer. The β gate loop (GL), which lies across the RNAP cleft from the tip of the clamp (Fig. 1), has been identified as an element that restricts the entry of the duplex promoter DNA into the narrow active site cleft (4), allowing only a single strand of DNA to pass through. This model posited that the DNA stands must separate outside the cleft before entry into RNAP, whereas footprinting studies demonstrated that the promoter DNA enters the active site cleft before it is opened (5). This controversy was a subject of an intense debate until a recent study revealed that the...

show abstract

Section: Resultssupporting

confidence: 81%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes

NandyMazumdar

Nedialkov

Svetlov

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…1, center) (26 -29). Furthermore, depending on the secondary RNA structure, the RNA 3Ј-end in the paused complex (corresponding to the third nucleotide from the RNA 3Ј-end in TEC-3p) was shown to fluctuate between distorted post-translocated and frayed pre-translocated conformations, both of which are unfavorable for RNA cleavage (26,28,39). We, therefore, propose that the paused RNAP conformation is preserved in TEC3p, which is based on the hisP sequence, thus making the target phosphodiester bond inaccessible for the TL action.…”

Section: Discussionmentioning

confidence: 99%

Conserved functions of the trigger loop and Gre factors in RNA cleavage by bacterial RNA polymerases

Miropolskaya

Esyunina

Kulbachinskiy

2017

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

RNA cleavage by RNA polymerase (RNAP) is the central step in co-transcriptional RNA proofreading. Bacterial RNAPs were proposed to rely on the same mobile element of the active site, the trigger loop (TL), for both nucleotide addition and RNA cleavage. RNA cleavage can also be stimulated by universal Gre factors, which should replace the TL to get access to the RNAP active site. The contributions of the TL and Gre factors to RNA cleavage reportedly vary between RNAPs from different bacterial species and, probably, different types of transcription complexes. Here, by comparing RNAPs from Escherichia coli, Deinococcus radiodurans, and Thermus aquaticus, we show that the functions of the TL and Gre factors in RNA cleavage are conserved in various species, with important variations that may be related to extremophilic adaptation. Deletions of the TL strongly impair intrinsic RNA cleavage by all three RNAPs and eliminate the interspecies differences in the reaction rates. GreA factors activate RNA cleavage by wild-type RNAPs to similar levels. The rates of GreA-dependent cleavage are lower for ⌬TL RNAP variants, suggesting that the TL contributes to the Gre function. Finally, neither the TL nor GreA can efficiently activate RNA cleavage in certain types of backtracked transcription complexes, suggesting that these complexes adopt a catalytically inactive conformation probably important for transcription regulation.The RNA cleavage reaction is thought to play a crucial role in correction of transcriptional errors during RNA synthesis by RNAP 2 (1-4). Because this reaction is performed by the same active site of RNAP as nucleotide addition, RNAP must "backtrack" along the DNA template after nucleotide misincorporation to allow cleavage of internal phosphodiester bonds in RNA. Long backtracking events can result in permanent stalling of RNAP in the backtracked state, and RNA cleavage also serves for reactivation of such "arrested" transcription elongation complexes (TECs) (5-7). After backtracking, the RNA 3Ј-end is accommodated within the secondary RNAP channel, which normally serves for NTP entry (Fig. 1). Both nucleotide addition and RNA cleavage require two divalent metal ions (usually Mg 2ϩ ) bound in the RNAP active site, which coordinate the reaction substrates (3,8). In addition to metal ions, several other factors were shown to contribute to RNA cleavage. In particular, the reaction is greatly stimulated by RNA nucleotide at position ϩ2 relative to the active site through its direct interactions with the second metal ion and the attacking water molecule (1). The ϩ2 nucleotide was also proposed to stimulate the second metal ion binding by changing the geometry of the active site residues involved in metal chelation ("active site tuning") (3). Recent crystallographic analysis of a bacterial backtracked TEC revealed that the ϩ2 RNA nucleotide is accommodated in an evolutionary conserved RNAP cavity ("proofreading site"), which facilitates catalysis (Fig. 1, left) (9). Similarly, interactions of the ϩ2 nucleotide i...

show abstract

“…The CRE motif is composed of two discontinuous polypeptide segments of the RpoB ␤ subunit (positions 440 to 455 and 535 to 555 in E. coli coordinates) (88,89,91). This motif therefore flanks domain I of region D, comprising the rifampin pocket fold (16).…”

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

View full text Add to dashboard Cite

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,...

show abstract

Mutations in the CRE pocket of bacterial RNA polymerase affect multiple steps of transcription

Cited by 28 publications

References 47 publications

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes

Conserved functions of the trigger loop and Gre factors in RNA cleavage by bacterial RNA polymerases

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

Contact Info

Product

Resources

About