RNA chain initiation by Escherichia coli RNA polymerase. Structural transitions of the enzyme in early ternary complexes

Krummel, Barbara; Chamberlin, Michael J.

doi:10.1021/bi00445a045

Cited by 225 publications

(193 citation statements)

References 46 publications

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“…For many years, it was widely believed that the transition to transcription elongation caused all 70 to be released from RNAP. A number of biochemical studies showed that ␤ and 70 could be purified from initiating but not from elongating RNAP complexes (2)(3)(4)(5). These data are consistent with structural studies of RNAP that suggest would be displaced from RNAP holoenzyme by RNA products of 12-14 nt in length (6).…”

supporting

confidence: 74%

Association of RNA polymerase with transcribed regions in Escherichia coli

Wade

Struhl

2004

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

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We examine the association of the ␤-, ␣-, and 70 -subunits of Escherichia coli RNA polymerase (RNAP) and the NusA elongation factor with transcribed regions in vivo by using chromatin immunoprecipitation. RNAP preferentially associates with the promoterproximal region of several operons, and this preference is particularly pronounced at the lexA-dinF promoter. When cells are grown in exponential phase, little or no 70 is associated with RNAP during early elongation. However, during stationary phase, 70 is retained in a fraction of elongating RNAP complexes throughout the melAB operon. In contrast, 70 is not observed in elongating RNAP complexes at the lacZYA operon during stationary phase. At both operons, NusA associates with RNAP during early elongation, and this association is greatly reduced during stationary phase. These observations suggest that in vivo association of 70 and NusA with elongating RNAP is regulated by growth conditions. transcription ͉ 70 ͉ NusA ͉ chromatin immunoprecipitation T he molecular understanding of transcriptional regulatory mechanisms in prokaryotes is well advanced, particularly in comparison to the understanding in eukaryotes. The association of RNA polymerase (RNAP) and associated proteins with transcribed regions of DNA has been studied extensively in vitro, and these biochemical experiments have provided great insight into the mechanisms of transcriptional initiation, elongation, and termination. In addition, there has been a great deal of genetic analysis, in which the transcriptional properties of mutant proteins and promoters have been assessed under various environmental conditions. Importantly, transcription in vitro is very efficient, occurring at rates comparable to those in living cells, and it faithfully mimics many aspects of transcription in vivo as defined by genetic analysis. However, there is very little work analyzing the association of RNAP and auxiliary proteins with transcribed regions in vivo.Escherichia coli RNA polymerase consists of five subunits: ␣ 2␤␤ Ј, but this core enzyme is unable to recognize promoters and accurately initiate transcription. RNAP associates with a number of accessory proteins during transcriptional initiation, elongation, and termination. During transcriptional initiation, core RNAP associates with a -subunit to form an RNAP holoenzyme. There are seven -subunits in E. coli, and each -subunit allows the RNAP holoenzyme to recognize a specific sequence at a subset of promoters (1). The predominant factor during exponential growth in rich media is 70 . For many years, it was widely believed that the transition to transcription elongation caused all 70 to be released from RNAP. A number of biochemical studies showed that ␤ and 70 could be purified from initiating but not from elongating RNAP complexes (2-5). These data are consistent with structural studies of RNAP that suggest would be displaced from RNAP holoenzyme by RNA products of 12-14 nt in length (6). However, more recent in vitro studies suggest that 70 can remain associated...

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supporting

confidence: 74%

Association of RNA polymerase with transcribed regions in Escherichia coli

Wade

Struhl

2004

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

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“…Such a model is not easily reconciled with bulk footprinting data which suggest that the abortive initiation process results from an inability of RNAP to break its promoter contacts (54)(55)(56). These observations led Straney and Crothers to propose that the energy required to break free of the promoter might be somehow stored in a "stressed intermediate," and that abortive initiation was a consequence of this energy not being used productively (55).…”

Section: Abortive Initiationmentioning

confidence: 79%

Single-Molecule Studies of RNA Polymerase: Motoring Along

Herbert

Greenleaf

Block

2008

Annu. Rev. Biochem.

187

141

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Single-molecule techniques have advanced our understanding of transcription by RNA polymerase. A new arsenal of approaches, including single-molecule fluorescence, atomic-force microscopy, magnetic tweezers, and optical traps have been employed to probe the many facets of the transcription cycle. These approaches supply fresh insights into the means by which RNA polymerase identifies a promoter; initiates transcription, translocates and pauses along the DNA template, proofreads errors, and ultimately terminates transcription. Results from single-molecule experiments complement knowledge gained from biochemical and genetic assays by facilitating the observation of states that are otherwise obscured by ensemble averaging, such as those resulting from heterogeneity in molecular structure, elongation rate, or pause propensity. Most studies to date have been performed with bacterial RNA polymerase, but work is also being carried out with eukaryotic polymerase (Pol II) and single-subunit polymerases from bacteriophages. We discuss recent progress achieved by single-molecule studies, highlighting some of the unresolved questions and ongoing debates.

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“…In general, transcription complexes stalled near the promoter (within the first 8 -10 bases) are much less stable than those stalled very distant from the promoter (1)(2)(3)(4). The transition between initiation and elongation is a complex process, involving release of stable promoter contacts and rearrangement of the protein and the nucleic acid (5-7).…”

mentioning

confidence: 99%

Observed Instability of T7 RNA Polymerase Elongation Complexes Can Be Dominated by Collision-induced “Bumping”

Zhou

Martin

2006

Journal of Biological Chemistry

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T7 RNA polymerase elongates RNA at a relatively high rate and can displace many tightly bound protein-DNA complexes. Despite these properties, measurements of the stability of stalled elongation complexes have shown lifetimes that are much shorter than those of the multisubunit RNA polymerases. In this work, we demonstrate that the apparent instability of stalled complexes actually arises from the action of trailing RNA polymerases (traveling in the same direction) displacing the stalled complex. Moreover, the instability caused by collision between two polymerases is position dependent. A second polymerase is blocked from promoter binding when a leading complex is stalled 12 bp or less from the promoter. The trailing complex can bind and make abortive transcripts when the leading complex is between 12 and 20 bp from the promoter, but it cannot displace the first complex since it is in a unstable initiation conformation. Only when the leading complex is stalled more than 20 bp away from the promoter can a second polymerase bind, initiate, and displace the leading complex.The process of transcription is characterized by distinct phases: initiation, elongation, and termination, at the simplest. In general, transcription complexes stalled near the promoter (within the first 8 -10 bases) are much less stable than those stalled very distant from the promoter (1-4). The transition between initiation and elongation is a complex process, involving release of stable promoter contacts and rearrangement of the protein and the nucleic acid (5-7). Consequently, it is not surprising that such complexes are relatively unstable. Since the released transcripts are relatively short, there should not be a large cellular price to inefficiency at this stage. In contrast, elongation complexes distant from the promoter must be very stable to productively synthesize long transcripts.The single subunit RNA polymerase from bacteriophage T7 presents an ideal model system in which to study transcription fundamentals. The enzyme is highly specific for a small 22-bp promoter and exhibits all the basic steps characteristic of the more complex multisubunit RNA polymerases (8, 9). As with the latter, initially transcribing complexes, with transcripts up to about 8 nucleotides, are particularly unstable and abortive products are released with frequency (1, 2, 10). Recent structural and biochemical studies indicate that a dramatic transition occurs on translocation beyond position ϩ8 and, while the enzyme-DNA-RNA ternary complex is relatively unstable before the transition, the complex becomes much more stable as the complex enters into the elongation stage (5)(6)(7)(11)(12)(13)(14).A previous study found that the apparent stability of complexes stepping away from the promoter follows an unexpected pattern: complexes stalled at positions ϩ10 and ϩ14 were observed to be more stable than complexes stalled at position ϩ22 (15). This result implied special properties for complexes recently clear of the promoter and suggested that the transition to el...

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RNA chain initiation by Escherichia coli RNA polymerase. Structural transitions of the enzyme in early ternary complexes

Cited by 225 publications

References 46 publications

Association of RNA polymerase with transcribed regions in Escherichia coli

Association of RNA polymerase with transcribed regions in Escherichia coli

Single-Molecule Studies of RNA Polymerase: Motoring Along

Observed Instability of T7 RNA Polymerase Elongation Complexes Can Be Dominated by Collision-induced “Bumping”

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