Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution

Vassylyev, Dmitry G.; Sekine, S.; Laptenko, Oleg; Lee, Jookyung; Vassylyeva, Marina N.; Borukhov, Sergei; Yokoyama, Shigeyuki

doi:10.1038/nature752

Cited by 709 publications

(701 citation statements)

References 45 publications

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“…4). In addition, the supposition that the P504L and S506F mutations in 70 alter and disrupt the normal path of region 3.2 with respect to its normal disposition within the RNA exit channel is supported by the physical defects displayed by these mutant 70 s. The loss or "loosening" of the interaction between the highly negatively charged region 3.2 with the highly positively charged RNA exit channel (40,41) would be expected to reduce 70 -core affinity which in fact is what has been observed (24).…”

Section: Structural Basis and Mechanism Of Mutant 70mentioning

confidence: 91%

“…The predicted path of the region 3 peptide that links domains 2 and 4 (referred to as the "Linker Domain") first descends into the enzyme crevice toward the active site and then makes a "hairpin turn" away from the active site to lay down the remainder of acidic region 3.2 into the path of the basic RNA exit channel. The presence of region 3.2 in the RNA exit channel is conjectured to pose steric clash between the advancing nascent RNA and the 70 protein, causing abortive release of the RNA (40,41).…”

Section: Structural Basis and Mechanism Of Mutant 70mentioning

confidence: 99%

“…Overall the subunit appears to be composed entirely of ␣-helices connected by either turns or loops (41), and the two 70 mutations we examined are located, by structural analogy, at the end of the 11th ␣-helix in the T. thermophilus holoenzyme structure (41). By using the SOPMA secondary structure prediction algorithm, both mutations are predicted to extend the ␣-helical potential of the 11th helix beyond proline 504 to position 510 where the next proline residue occurs.…”

Section: Structural Basis and Mechanism Of Mutant 70mentioning

confidence: 99%

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Changes in Conserved Region 3 of Escherichia coliς70 Reduce Abortive Transcription and Enhance Promoter Escape

Cashel

Hsu

Hernandez

2003

Journal of Biological Chemistry

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Mutations within the Escherichia coli rpoD gene encoding amino acid substitutions in conserved region 3 of the 70 subunit of E. coli RNA polymerase restore normal stress responsiveness to strains devoid of the stress alarmone, guanosine-3 ,5 -(bis)pyrophosphate (ppGpp). The presence of a mutant protein, either 70 (P504L) or 70 (S506F), suppresses the physiological defects in strains devoid of ppGpp. In vitro, when reconstituted into RNA polymerase holoenzyme, these mutants confer unique transcriptional properties, namely they reduce the probabilities of forming abortive RNAs. Here we investigated the behavior of these mutant enzymes during transcription of the highly abortive cellular promoter, gal P2. No differences between mutant and wildtype enzymes were observed prior to and including open complex formation. Remarkably, the mutant enzymes produced drastically reduced levels of gal P2 abortive RNAs and increased production of full-length gal P2 RNAs relative to the wild-type enzyme, leading to greatly reduced ratios of abortive to productive RNAs. These results are attributed mainly to a decreased formation of unproductive initial transcribing complexes with the mutant polymerases and increased rates of promoter escape. Altered transcription properties of these mutant polymerases arise from an alternative structure of the 70 region 3.2 segment that permits efficient positioning of the nascent RNA into the RNA exit channel displacing and facilitating release.The steps in transcription initiation performed by all DNAdependent RNA polymerases are promoter binding, DNA strand melting, RNA chain initiation and nascent RNA chain formation, and finally escape from the promoter sequences. Failure to escape from the promoter results in the production of aborted RNA transcripts and reiterative cycling through the open complex (1-4). Promoter escape appears to occur concomitantly with the release of 70 factor following the synthesis of a specific length of RNA and the conversion of the unstable initial transcribing complex to a stable elongating complex (5-8). Depending on the promoter, abortive RNA synthesis occurs to different degrees and can occur extensively enough that it becomes rate-limiting for the synthesis of productive RNAs (2, 9). Important determinants of abortive initiation are contained along the region of melted DNA within the transcription complex encompassing the non-transcribed and the initial transcribed sequences (ITS). 1 Changes in the composition of the ITS can alter the extent of productive RNA synthesis (10) and cause dramatic variation in the total number of abortive events and also alter abortive probabilities from position to position within the ITS (11). Base residue changes specifically in the non-template strand of the Ϫ10 promoter region, which weaken or strengthen open complex stability, can lead to decreased or increased abortive probabilities, respectively (12). Several examples of regulation at the level of promoter escape have been reported. The phage ⌽29 promoter A2c was repressed b...

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Section: Structural Basis and Mechanism Of Mutant 70mentioning

confidence: 91%

Section: Structural Basis and Mechanism Of Mutant 70mentioning

confidence: 99%

Section: Structural Basis and Mechanism Of Mutant 70mentioning

confidence: 99%

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Changes in Conserved Region 3 of Escherichia coliς70 Reduce Abortive Transcription and Enhance Promoter Escape

Cashel

Hsu

Hernandez

2003

Journal of Biological Chemistry

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“…The transcription process is carried out by RNA polymerase (RNAP) (1-3), a multisubunit molecular motor, the basic structure of which is conserved from bacteria to eukaryotes (4). Over the past decade a great deal has been learned about the structure of RNAP, particularly in the context of yeast (5,6), thermophilic bacteria (7,8), and Escherichia coli (9)(10)(11)(12). Given the high degree of structural conservation of RNAP, and the synthesis of structural, biochemical, and kinetic information from bacteria and yeast, we are able to begin building mechanistic models of transcription valid across species.…”

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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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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“…6 In the bacterial transcription initiation complex, a region of the initiation factor contacts or closely approaches the transcription-bubble region near the transcription start site: region 3.2 ( R3.2, also known as the 3 -4 linker (18, 19)). R3.2 consists of two sub-regions: an extended segment that binds within the RNAP RNA exit channel (18,19,57) and a hairpin loop that binds within the RNAP active-center cleft between the two strands of the melted transcription bubble, near the transcription start site (18,19). Deletion of the R3.2 hairpin loop affects transcription-initiation NTP concentration dependence and transcription-initiation efficiency (18,62).…”

Section: Ment Both In Thementioning

confidence: 99%

Transcription Factor B Contacts Promoter DNA Near the Transcription Start Site of the Archaeal Transcription Initiation Complex

Renfrow

Naryshkin

Lewis

et al. 2004

Journal of Biological Chemistry

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Transcription initiation in all three domains of life requires the assembly of large multiprotein complexes at DNA promoters before RNA polymerase (RNAP)-catalyzed transcript synthesis. Core RNAP subunits show homology among the three domains of life, and recent structural information supports this homology. General transcription factors are required for productive transcription initiation complex formation. The archaeal general transcription factors TATA-element-binding protein (TBP), which mediates promoter recognition, and transcription factor B (TFB), which mediates recruitment of RNAP, show extensive homology to eukaryal TBP and TFIIB. Crystallographic information is becoming available for fragments of transcription initiation complexes (e.g. RNAP, TBP-TFB-DNA, TBP-TFIIB-DNA), but understanding the molecular topography of complete initiation complexes still requires biochemical and biophysical characterization of protein-protein and protein-DNA interactions. In published work, systematic site-specific protein-DNA photocrosslinking has been used to define positions of RNAP subunits and general transcription factors in bacterial and eukaryal initiation complexes. In this work, we have used systematic site-specific protein-DNA photocrosslinking to define positions of RNAP subunits and general transcription factors in an archaeal initiation complex. Employing a set of 41 derivatized DNA fragments, each having a phenyl azide photoactivable crosslinking agent incorporated at a single, defined site within positions ؊40 to ؉1 of the gdh promoter of the hyperthermophilic marine archaea, Pyrococcus furiosus (Pf), we have determined the locations of PfRNAP subunits PfTBP and PfTFB relative to promoter DNA. The resulting topographical information supports the striking homology with the eukaryal initiation complex and permits one major new conclusion, which is that PfTFB interacts with promoter DNA not only in the TATA-element region but also in the transcription-bubble region, near the transcription start site. Comparison with crystallographic information implicates the PfTFB N-terminal domain in the interaction with the transcription-bubble region. The results are discussed in relation to the known effects of substitutions in the TFB and TFIIB N-terminal domains on transcription initiation and transcription start-site selection.Extensive structural and biochemical characterization of transcription initiation complexes has revealed similarity of transcription systems across the three domains of life (1-4). Transcription initiation in archaea closely resembles eukaryal class II transcription initiation and is mediated by a single RNA polymerase (RNAP) 1 and two general transcription factors, TATA-element binding protein (TBP) and transcription factor B (TFB) (5, 6). In vitro transcription initiation has been demonstrated with RNAP, TBP, and TFB in Pyrococcus (7-10), Methanococcus (11), Methanosarcina (12), and Sulfolobus (13) cell-free transcription systems.Recent crystallographic structures of bacterial RNAP and eukary...

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Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution

Cited by 709 publications

References 45 publications

Changes in Conserved Region 3 of Escherichia coliς70 Reduce Abortive Transcription and Enhance Promoter Escape

Changes in Conserved Region 3 of Escherichia coliς70 Reduce Abortive Transcription and Enhance Promoter Escape

Thermodynamic and kinetic modeling of transcriptional pausing

Transcription Factor B Contacts Promoter DNA Near the Transcription Start Site of the Archaeal Transcription Initiation Complex

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