Regulation of RNA Polymerase through the Secondary Channel

Nickels, Bryce E.; Hochschild, Ann

doi:10.1016/j.cell.2004.07.021

Cited by 57 publications

(42 citation statements)

References 19 publications

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“…In particular, we consider the possibility of predicting pause positions where RNAP halts either reversibly (pauses) or irreversibly (arrests) (13). Pauses, which are characterized by highly variable durations and efficiencies (3), are an ubiquitous aspect of transcription elongation and are known to play regulatory roles particularly in synchronizing transcription with other biological processes, such as translation in bacteria (14), factor-dependent and factor-independent termination (15,16), and interactions with regulatory proteins (17). Even though pauses are not associated with a consensus sequence, pause positions along the template are strongly sequence-dependent.…”

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confidence: 99%

Thermodynamic and kinetic modeling of transcriptional pausing

Tadigotla

Maoiléidigh

Sengupta

et al. 2006

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

106

185

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We present a statistical mechanics approach for the prediction of backtracked pauses in bacterial transcription elongation derived from structural models of the transcription elongation complex (EC). Our algorithm is based on the thermodynamic stability of the EC along the DNA template calculated from the sequence-dependent free energy of DNA–DNA, DNA–RNA, and RNA–RNA base pairing associated with ( i ) the translocational and size fluctuations of the transcription bubble; ( ii ) changes in the associated DNA–RNA hybrid; and ( iii ) changes in the cotranscriptional RNA secondary structure upstream of the RNA exit channel. The calculations involve no adjustable parameters except for a cutoff used to discriminate paused from nonpaused complexes. When applied to 100 experimental pauses in transcription elongation by Escherichia coli RNA polymerase on 10 DNA templates, the approach produces statistically significant results. We also present a kinetic model for the rate of recovery of backtracked paused complexes. A crucial ingredient of our model is the incorporation of kinetic barriers to backtracking resulting from steric clashes of EC with the cotranscriptionally generated RNA secondary structure, an aspect not included explicitly in previous attempts at modeling the transcription elongation process.

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mentioning

confidence: 99%

Thermodynamic and kinetic modeling of transcriptional pausing

Tadigotla

Maoiléidigh

Sengupta

et al. 2006

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

106

185

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“…12). We suggest that detection of defects in regulation in ⌬dksA strains could potentially be compromised by compensatory effects of other DksA-like proteins, such as GreA and GreB, that can inhabit the secondary channel of RNAP (38,39) or by other compensatory mechanisms, especially given the extended time intervals inherent in steady-state assays. Fig.…”

Section: Effects Of Dksa On Positive Regulation Of Amino Acid Promotementioning

confidence: 99%

DksA potentiates direct activation of amino acid promoters by ppGpp

Paul

Berkmen

Gourse

2005

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

299

414

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Amino acid starvation in Escherichia coli results in a spectrum of changes in gene expression, including inhibition of rRNA and tRNA promoters and activation of certain promoters for amino acid biosynthesis and transport. The unusual nucleotide ppGpp plays an important role in both negative and positive regulation. Previously, we and others suggested that positive effects of ppGpp might be indirect, resulting from the inhibition of rRNA transcription and, thus, liberation of RNA polymerase for binding to other promoters. Recently, we showed that DksA binds to RNA polymerase and greatly enhances direct effects of ppGpp on the negative control of rRNA promoters. This conclusion prompted us to reevaluate whether ppGpp might also have a direct role in positive control. We show here that ppGpp greatly increases the rate of transcription initiation from amino acid promoters in a purified system but only when DksA is present. Activation occurs by stimulation of the rate of an isomerization step on the pathway to open complex formation. Consistent with the model that ppGpp͞ DksA stimulates amino acid promoters both directly and indirectly in vivo, cells lacking dksA fail to activate transcription from the hisG promoter after amino acid starvation. Our results illustrate how transcription factors can positively regulate transcription initiation without binding DNA, demonstrate that dksA directly affects promoters in addition to those for rRNA, and suggest that some of the pleiotropic effects previously associated with dksA might be ascribable to direct effects of dksA on promoters involved in a wide variety of cellular functions.RNA polymerase ͉ rRNA transcription ͉ transcription initiation ͉ amino acid biosynthesis ͉ stringent response A mino acid starvation in Escherichia coli results in global changes in gene expression called the ''stringent response'' (reviewed in ref. 1). This regulatory response is initiated by entry of uncharged tRNAs into the A site of the ribosome and activation of the ribosome-associated RelA protein to synthesize the ''alarmone'' ppGpp (used here to refer to both guanosine 5Ј-diphosphate 3Ј-diphosphate and its pentaphosphate precursor). During starvation for amino acids, expression of stable RNA (rRNA and tRNA) is inhibited, whereas expression of enzymes for amino acid biosynthesis and transport is induced. These negative and positive responses are both relA dependent (e.g., refs. 2-6).ppGpp directly and specifically inhibits the initiation of transcription from stable RNA promoters in vivo and in vitro (7)(8)(9)(10)(11)(12). Although the details of the mechanism by which ppGpp exerts its effects on transcription initiation are still ill-defined, it has been shown that ppGpp decreases the lifetimes of competitorresistant complexes between RNA polymerase (RNAP) and all promoters that have been examined (12). The complex formed by RNAP at rRNA promoters is intrinsically short-lived, and ppGpp further decreases the lifetime of this complex. Thus, we have proposed that ppGpp shifts the equilibriu...

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“…Escherichia coli possesses two highly homologous Gre factors: GreA and GreB. A structural study on the RNAP-GreB complex further revealed that CTD binds to the rim of the secondary channel of RNAP through which substrate nucleoside triphosphates for RNA synthesis enter the catalytic site (18,25,38), while NTD extends into the secondary channel and the tip reaches the catalytic center (28). Two acidic residues, D41 and E44, located at the tip of NTD, are conserved in Gre factors, including those of Bacillus subtilis, and are proposed to assist RNAP function by coordinating the Mg 2ϩ ion and water molecule required for catalysis of RNA hydrolysis (20,28,31).…”

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confidence: 99%

Transcription Factor GreA Contributes to Resolving Promoter-Proximal Pausing of RNA Polymerase in Bacillus subtilis Cells

et al. 2011

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Bacterial Gre factors associate with RNA polymerase (RNAP) and stimulate intrinsic cleavage of the nascent transcript at the active site of RNAP. Biochemical and genetic studies to date have shown that Escherichia coli Gre factors prevent transcriptional arrest during elongation and enhance transcription fidelity. Furthermore, Gre factors participate in the stimulation of promoter escape and the suppression of promoter-proximal pausing during the beginning of RNA synthesis in E. coli. Although Gre factors are conserved in general bacteria, limited functional studies have been performed in bacteria other than E. coli. In this investigation, ChAP-chip analysis (chromatin affinity precipitation coupled with DNA microarray) was conducted to visualize the distribution of Bacillus subtilis GreA on the chromosome and to determine the effects of GreA inactivation on core RNAP trafficking. Our data show that GreA is uniformly distributed in the transcribed region from the promoter to coding region with core RNAP, and its inactivation induces RNAP accumulation at many promoter or promoter-proximal regions. Based on these findings, we propose that GreA would constantly associate with core RNAP during transcriptional initiation and elongation and resolves its stalling at promoter or promoter-proximal regions, thus contributing to the even distribution of RNAP along the promoter and coding regions in B. subtilis cells.Bacterial Gre factors associate with RNA polymerase (RNAP) and stimulate intrinsic cleavage of the nascent transcript at the active site of the enzyme (12). In eukaryotic cells, the transcription factor, TFIIS, exerts similar activity (9), indicating that Gre function is evolutionarily conserved in multisubunit RNAPs (9). Gre factors consist of an N-terminal extended coiled-coil domain (NTD) and C-terminal globular domain (CTD) (19,32). Escherichia coli possesses two highly homologous Gre factors: GreA and GreB. A structural study on the RNAP-GreB complex further revealed that CTD binds to the rim of the secondary channel of RNAP through which substrate nucleoside triphosphates for RNA synthesis enter the catalytic site (18,25,38), while NTD extends into the secondary channel and the tip reaches the catalytic center (28). Two acidic residues, D41 and E44, located at the tip of NTD, are conserved in Gre factors, including those of Bacillus subtilis, and are proposed to assist RNAP function by coordinating the Mg 2ϩ ion and water molecule required for catalysis of RNA hydrolysis (20,28,31).During the elongation process of transcription, roadblocks generated by DNA-binding proteins or specific DNA sequences induce RNAP to slide backward along the template (backtrack), resulting in extrusion of the 3Ј terminus of nascent RNA through the RNAP secondary channel (9). Several biochemical and genetic studies have confirmed that the Gre factor facilitates endonucleolytic cleavage of extruded RNA to generate a new terminus that can be extended by RNAP, thus preventing transcription arrest during elongation and enhancing t...

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Regulation of RNA Polymerase through the Secondary Channel

Cited by 57 publications

References 19 publications

Thermodynamic and kinetic modeling of transcriptional pausing

Thermodynamic and kinetic modeling of transcriptional pausing

DksA potentiates direct activation of amino acid promoters by ppGpp

Transcription Factor GreA Contributes to Resolving Promoter-Proximal Pausing of RNA Polymerase in Bacillus subtilis Cells

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