Prevalence of RNA polymerase stalling at <i>Escherichia coli</i> promoters after open complex formation

Hatoum, Asma; Roberts, Jeffrey W.

doi:10.1111/j.1365-2958.2008.06138.x

Cited by 60 publications

(83 citation statements)

References 42 publications

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“…Furthermore, the same module of s 70 that contacts the promoter À10 element during transcription initiation (s 70 region 2) contacts the À10-like element in the initial transcribed region during early elongation pausing (Ring et al 1996). Similar À10-like initial transcribed region DNA sequence elements have been shown to induce s 70 -dependent pausing in the context of the late promoters of other lambdoid phages (Ring et al 1996) and in the context of several E. coli promoters, including the lac, tnaA, and cspD promoters (Brodolin et al 2004;Nickels et al 2004;Hatoum and Roberts 2008). Furthermore, both bioinformatics analysis and a candidate-based approach suggest that ;20% of all E. coli promoters may carry s…”

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confidence: 98%

“…s 70 -dependent pausing has been shown to affect the production of full-length transcripts, presumably by modulating RNAP density within the transcription unit (see also Fig. 4D; Hatoum and Roberts 2008). Such effects on RNAP density within a transcription unit could, in principle, affect the overall elongation rate and modulate the responsiveness of RNAP to termination signals by influencing the extent of RNAP ''cooperation'' (Epshtein The 4132 transcription start sites were identified by Cho et al (2009).…”

Section: Functional Roles For S 70 During Transcription Elongationmentioning

confidence: 99%

“…Interestingly, promoter-distal s 70 -dependent pausing has been proposed to occur at a cluster of sites extending as far as ;100 bp downstream from the transcription start site of the E. coli tnaA promoter, which also carries a s 70 -dependent pause element in its initial transcribed region (Hatoum and Roberts 2008). Our findings suggest that the observed promoter-distal pausing in the tnaA transcription unit may be influenced by the presence of the s 70 -dependent pause element in the initial transcribed region.…”

Section: Functional Roles For S 70 During Transcription Elongationmentioning

confidence: 99%

“…-dependent pause-inducing sequence elements in their initial transcribed regions (Nickels et al 2004;Hatoum and Roberts 2008).…”

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confidence: 99%

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Initial transcribed region sequences influence the composition and functional properties of the bacterial elongation complex

Deighan¹,

Pukhrambam²,

Nickels³

et al. 2011

Genes Dev.

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The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme (a 2 bb'v) in complex with a s factor that is essential for promoter recognition and transcription initiation. During early elongation, the stability of interactions between s and the remainder of the transcription complex decreases. Nevertheless, there is no mechanistic requirement for release of s upon the transition to elongation. Furthermore, s can remain associated with RNAP during transcription elongation and influence regulatory events that occur during transcription elongation. Here we demonstrate that promoter-like DNA sequence elements within the initial transcribed region that are known to induce early elongation pausing through sequence-specific interactions with s also function to increase the s content of downstream elongation complexes. Our findings establish s-dependent pausing as a mechanism by which initial transcribed region sequences can influence the composition and functional properties of the transcription elongation complex over distances of at least 700 base pairs.[Keywords: RNA polymerase; s factor; transcription initiation; promoter-proximal pausing; transcription elongation complex] Supplemental material is available for this article. The s subunit of bacterial RNA polymerase (RNAP) is required for promoter-specific transcription initiation (Gross et al. 1998). When complexed with the RNAP core enzyme (subunit structure a 2 bb9v), different s factors specify the recognition of different classes of promoters (Gruber and Gross 2003). The primary s factor in Escherichia coli, s 70 , typically directs transcription initiation from promoters defined by two conserved hexameric DNA sequence elements, termed the À10 and À35 elements for their relationship to the transcription start site (position +1). During the transition from transcription initiation to transcription elongation, the growth of the nascent RNA destabilizes the interaction between s 70 and the RNAP core enzyme (Mekler et al. 2002;Murakami et al. 2002;Vassylyev et al. 2002;Nickels et al. 2005), but the complete release of s 70 is not required for entry into the elongation phase of transcription (Ring et al. 1996;Bar-Nahum and Nudler 2001;Mukhopadhyay et al. 2001;Mooney et al. 2005). Thus, although historically defined as an initiation factor, s 70 can also remain associated with the transcription elongation complex and influence the transcription process during elongation. A functional role for s 70 during elongation was first established in the context of bacteriophage l late gene transcription (for review, see Roberts et al. 1998). Specifically, the expression of the phage late genes under the control of the lQ anti-terminator protein depends on a s 70 -dependent pause that occurs during early elongation, shortly after the s 70 -containing RNAP holoenzyme has escaped the late promoter P R9 . Formation of this paused early elongation complex, which contains a stably bound nascent RNA that is 16 or 17 nucleotides (nt) in length, depends on an int...

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mentioning

confidence: 98%

Section: Functional Roles For S 70 During Transcription Elongationmentioning

confidence: 99%

Section: Functional Roles For S 70 During Transcription Elongationmentioning

confidence: 99%

“…-dependent pause-inducing sequence elements in their initial transcribed regions (Nickels et al 2004;Hatoum and Roberts 2008).…”

mentioning

confidence: 99%

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Initial transcribed region sequences influence the composition and functional properties of the bacterial elongation complex

Deighan¹,

Pukhrambam²,

Nickels³

et al. 2011

Genes Dev.

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“…Similarly, the transition from abortive initiation to processive elongation is an essential event of transcription that often dictates the rate at which the full-length RNA is made (3)(4)(5)(6)(7)(8)(9). During the abortive initiation phase, the RNAP maintains contacts with the promoter and catalyzes RNA polymerization by scrunching the template DNA (10)(11)(12)(13).…”

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confidence: 99%

Real-time observation of the transition from transcription initiation to elongation of the RNA polymerase

Tang¹,

Roy²,

Bandwar³

et al. 2009

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

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The transition from initiation to elongation of the RNA polymerase (RNAP) is an important stage of transcription that often limits the production of the full-length RNA. Little is known about the RNAP transition kinetics and the steps that dictate the transition rate, because of the challenge in monitoring subpopulations of the transient and heterogeneous transcribing complexes in rapid and real time. Here, we have dissected the complete transcription initiation pathway of T7 RNAP by using kinetic modeling of RNA synthesis and by determining the initiation (IC) to elongation (EC) transition kinetics at each RNA polymerization step using single-molecule and stoppedflow FRET methods. We show that the conversion of IC to EC in T7 RNAP consensus promoter occurs only after 8-to 12-nt synthesis, and the 12-nt synthesis represents a critical juncture in the transcriptional initiation pathway when EC formation is most efficient. We show that the slow steps of transcription initiation, including DNA scrunching/ RNAP-promoter rotational changes during 5-to 8-nt synthesis, not the major conformational changes, dictate the overall rate of EC formation in T7 RNAP and represent key steps that regulate the synthesis of full-length RNA.abortive synthesis ͉ FRET ͉ rate-limiting ͉ T7 RNA polymerase ͉ transcription transition T he recruitment of RNA polymerase (RNAP) to the promoter and isomerization to a competent open complex are important regulatory steps in gene transcription (1, 2). Similarly, the transition from abortive initiation to processive elongation is an essential event of transcription that often dictates the rate at which the full-length RNA is made (3-9). During the abortive initiation phase, the RNAP maintains contacts with the promoter and catalyzes RNA polymerization by scrunching the template DNA (10-13). After a certain length of RNA is made, the RNAP switches transcription from promoter-specific to promoter-independent and processive RNA synthesis. Single-subunit and multisubunit RNAPs switch from the initiation complex (IC) to the elongation complex (EC) by undergoing specific structural rearrangements. For example, the single-subunit T7 RNAP protein undergoes major refolding of its N-terminal domain during EC transition that releases promoter contacts and results in the formation of RNA channel (14,15). The bacterial RNAP makes this transition by releasing the sigma factor from the promoter (7,(16)(17)(18)(19), and in eukaryotic RNAP II, this event is marked by the removal of several transcription factors, including TFIIB, TFIIE, TFIIF, and TFIIH (20). Although the initiation stage and the transition from IC to EC are major points of regulation that ultimately control gene expression, the rate-limiting steps governing these multistep processes and the kinetics of transition have not been elucidated.The challenge in studying the kinetic pathway of initiation and the IC-to-EC transition has been the transient and heterogeneous nature of the initially transcribing complexes. Studies of the transient heterogen...

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Nucleotide Analogues as Probes for DNA and RNA Polymerases

Kuchta

2010

CP Chemical Biology

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Nucleotide analogues represent a major class of anti‐cancer and anti‐viral drugs, and provide an extremely powerful tool for dissecting the mechanisms of DNA and RNA polymerases. While the basic assays themselves are relatively straightforward, a key issue is to appropriately design the studies to answer the mechanistic question of interest. This unit addresses the major issues involved in designing these studies, and some of the potential difficulties that arise in interpreting the data. Examples are given for the type of analogues typically used, the experimental approaches with different polymerases, and issues with data interpretation. Curr. Protoc. Chem Biol. 2:111‐124. © 2010 by John Wiley & Sons, Inc.

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Prevalence of RNA polymerase stalling at Escherichia coli promoters after open complex formation

Cited by 60 publications

References 42 publications

Initial transcribed region sequences influence the composition and functional properties of the bacterial elongation complex

Initial transcribed region sequences influence the composition and functional properties of the bacterial elongation complex

Real-time observation of the transition from transcription initiation to elongation of the RNA polymerase

Nucleotide Analogues as Probes for DNA and RNA Polymerases

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