Nucleation of RNA Chain Formation by Escherichia coli DNA-dependent RNA Polymerase

Metzger, Willi; Schickor, Peter; Meier, Thomas; Werel, W.; Heumann, Hermann

doi:10.1006/jmbi.1993.1368

Cited by 53 publications

(60 citation statements)

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“…The presence of GTP and CTP would be expected to allow synthesis of the trinucleotide GGC (Fig. 4A), but in abortive initiation experiments (data not shown), some transcripts of 9 nucleotides or greater were present, indicating either that this nucleotide combination may be contaminated or that misincorporation occurs (23). In the presence of GTP, CTP, and ATP, most complexes were rifampin resistant, as might be expected (28) (Fig.…”

Section: Resultssupporting

confidence: 51%

“…There may be some differences between the nature of the initiated complex formed with E 54 and that of the complex formed with E may be stabilized by the process of abortive initiation (10,34). In contrast, whereas open complexes formed at the T7 A1 promoter are heparin resistant, the abortive transcription complex is heparin sensitive (23). Although our results strongly suggest that GTP stabilizes open complexes as a consequence of its role as the initiating nucleotide, we cannot rule out the possibility that NIFA in the presence of GTP reduces the dissociation rate of E 54 in open complexes via a direct protein-protein interaction, as has been observed for the catabolite gene activator protein-RNA polymerase association at the lac promoter (33).…”

Section: Discussionmentioning

confidence: 99%

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Transcriptional activation of the nitrogenase promoter in vitro: adenosine nucleotides are required for inhibition of NIFA activity by NIFL

et al. 1995

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The enhancer-binding protein NIFA is required for transcriptional activation of nif promoters by the alternative holoenzyme form of RNA polymerase, which contains the sigma factor 54 ( N ). NIFA hydrolyzes nucleoside triphosphates to catalyze the isomerization of closed promoter complexes to transcriptionally competent open complexes. The activity of NIFA is antagonized by the regulatory protein NIFL in response to oxygen and fixed nitrogen in vivo. We have investigated the requirement for nucleotides in the formation and stability of open promoter complexes by NIFA and inhibition of its activity by NIFL at the Klebsiella pneumoniae nifH promoter. Open complexes formed by 54 -containing RNA polymerase are considerably more stable to heparin challenge in the presence of GTP than in the presence of ATP. This differential stability is most probably a consequence of GTP being the initiating nucleotide at this promoter. Adenosine nucleosides are specifically required for Azotobacter vinelandii NIFL to inhibit open complex formation by native NIFA, and the nucleoside triphosphatase activity of NIFA is strongly inhibited by NIFL under these conditions. We propose a model in which NIFL modulates the activity of NIFA via an adenosine nucleotide switch.A distinct mechanism of transcriptional activation is observed among the family of prokaryotic enhancer-binding proteins which interact with the holoenzyme form of RNA polymerase containing the alternative sigma factor 54 (E 54 ) (19,26). The nitrogen fixation regulatory protein NIFA is a member of this family which binds to upstream activator sequences (UAS) and catalyzes the isomerization of closed promoter complexes to the open complex in a reaction which requires hydrolysis of a nucleoside triphosphate (25). Productive interactions between NIFA and E 54 are enabled by DNA loop formation, which is facilitated by the binding of integration host factor (IHF) (18,30). The amino acid sequence of NIFA conforms to the three-domain model for the structure of pneumoniae NIFA activates transcription in the absence of specific DNA binding and possesses nucleoside triphosphatase activity (6). In contrast to its K. pneumoniae counterpart, the native Azotobacter vinelandii NIFA protein has been purified in a soluble form, and its properties with respect to DNA binding and catalysis of open complex formation have been characterized in vitro (2).In both K. pneumoniae and A. vinelandii, the activity of NIFA is controlled by a second regulatory protein, NIFL, in response to the environmental effectors oxygen and fixed nitrogen (7,22). Although NIFL proteins show homology in their C-terminal domains to the histidine protein kinase family of two-component regulatory proteins (14), NIFL and NIFA appear to interact at stoichiometric levels (5, 15), and phosphotransfer between the two proteins has not been detected in vitro (2, 21). Moreover, although A. vinelandii NIFL shows greater homology to the canonical histidine protein kinases than does K. pneumoniae NIFL and contains a conserved...

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Section: Resultssupporting

confidence: 51%

Section: Discussionmentioning

confidence: 99%

Transcriptional activation of the nitrogenase promoter in vitro: adenosine nucleotides are required for inhibition of NIFA activity by NIFL

et al. 1995

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“…Proteins-FIS and RNA polymerase were isolated as described previously (36,37). Purified CRP was kindly provided by Dr. A.…”

Section: Methodsmentioning

confidence: 99%

CRP Modulates fis Transcription by Alternate Formation of Activating and Repressing Nucleoprotein Complexes

Nasser

Schneider

Travers

et al. 2001

Journal of Biological Chemistry

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The DNA architectural proteins FIS and CRP are global regulators of transcription in Escherichia coli involved in the adjustment of cellular metabolism to varying growth conditions. We have previously demonstrated that FIS modulates the expression of the crp gene by functioning as its transcriptional repressor. Here we show that in turn, CRP is required to maintain the growth phase pattern of fis expression. We demonstrate the existence of a divergent promoter in the fis regulatory region, which reduces transcription of the fis promoter. In the absence of FIS, CRP activates fis transcription, thereby displacing the polymerase from the divergent promoter, whereas together FIS and CRP synergistically repress fis gene expression. These results provide evidence for a direct cross-talk between global regulators of cellular transcription during the growth phase. This cross-talk is manifested in alternate formation of functional nucleoprotein complexes exerting either activating or repressing effects on transcription.The rapid reorganization of cellular metabolism in response to changing growth conditions is a hallmark of bacterial cells. These metabolic reorganizations involve global regulators, many of which serve as DNA architectural factors associated with bacterial chromatin (1, 2). The abundant DNA bending chromatin protein FIS is a global regulator of bacterial metabolism facilitating the adjustment of cells to rapid growth conditions (3-6). The expression of fis positively correlates with the richness of the medium (5), such that the amount of FIS protein produced is commensurate with the available metabolic potential. FIS increases the synthesis of stable RNA (tRNA and rRNA) species (7-9), adjusting the capacity of the translational machinery to changes in the nutritional supply. FIS is thought to activate the transcription of stable RNA genes by stabilizing local DNA architectures in their promoter regions (10). However, at many other gene promoters, including the fis promoter, FIS acts as a transcriptional repressor (11)(12)(13)(14)(15)(16)(17)(18).The transcription of the fis operon sharply increases on subculturing stationary cells in rich medium, then steeply decreases because of autoregulation by FIS, and ceases in late exponential phase (11,16). This pattern of expression during the growth phase is probably optimized, because constitutive fis expression negatively affects the survival of cells under stress conditions (19). The expression of fis is largely regulated at the transcriptional level without any significant contribution of fis mRNA decay rates (11,16,20). As expected for a gene involved in the modulation of cellular physiology, the expression of fis is subject to a plethora of control mechanisms. The activity of the fis promoter responds both to the growth ratedependent and stringent control systems (16). This latter abrogates stable RNA production on limitation of amino acid availability and is mediated by the nucleotide ppGpp (21). However, the role of ppGpp in the shut-off of fis expression...

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“…Failure to escape from the promoter results in the production of aborted RNA transcripts and reiterative cycling through the open complex (1)(2)(3)(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)(6)(7)(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).…”

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confidence: 99%

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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Nucleation of RNA Chain Formation by Escherichia coli DNA-dependent RNA Polymerase

Cited by 53 publications

References 0 publications

Transcriptional activation of the nitrogenase promoter in vitro: adenosine nucleotides are required for inhibition of NIFA activity by NIFL

Transcriptional activation of the nitrogenase promoter in vitro: adenosine nucleotides are required for inhibition of NIFA activity by NIFL

CRP Modulates fis Transcription by Alternate Formation of Activating and Repressing Nucleoprotein Complexes

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

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