rRNA Promoter Regulation by Nonoptimal Binding of σ Region 1.2: An Additional Recognition Element for RNA Polymerase

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110

183

SummaryEscherichia coli DksA, GreA, and GreB have similar structures and bind to the same location on RNA polymerase (RNAP), the secondary channel. We show that GreB can fulfill some roles of DksA in vitro, including shifting the promoter-open complex equilibrium in the dissociation direction, thus allowing rRNA promoters to respond to changes in the concentrations of ppGpp and NTPs. However, unlike deletion of the dksA gene, deletion of greB had no effect on rRNA promoters in vivo. We show that the apparent affinities of DksA and GreB for RNAP are similar, but the cellular concentration of GreB is much lower than that of DksA. When overexpressed and in the absence of competing GreA, GreB almost completely complemented the loss of dksA in control of rRNA expression, indicating its inability to regulate rRNA transcription in vivo results primarily from its low concentration. In contrast to GreB, the apparent affinity of GreA for RNAP was weaker than that of DksA, GreA only modestly affected rRNA promoters in vitro, and even when overexpressed GreA did not affect rRNA transcription in vivo. Thus, binding in the secondary channel is necessary but insufficient to explain the effect of DksA on rRNA transcription. Neither Gre factor was capable of fulfilling two other functions of DksA in transcription initiation: co-activation of amino acid biosynthetic gene promoters with ppGpp and compensation for the loss of the ω subunit of RNAP in the response of rRNA promoters to ppGpp. Our results provide important clues to the mechanisms of both negative and positive control of transcription initiation by DksA.

Section: Greb and Dksa Similarly Decrease The Half-life Of The Rnap-pmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Effects of DksA, GreA, and GreB on Transcription Initiation: Insights into the Mechanisms of Factors that Bind in the Secondary Channel of RNA Polymerase

Rutherford

Lemke

Vrentas

et al. 2007

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110

183

“…Promoters are recognized by the promoter specificity subunit (s) of the RNA polymerase holoenzyme through sequence-specific interactions of s domain 4 (bound to the flap of the RNAP b subunit) with the -35 motif, and domain 2 (bound to the RNAP b' subunit coiled coil) with the -10 motif. Additional interactions, of the two RNAP a subunit C-terminal domains (aCTDs) with DNA upstream of position ∼-40, and of other regions of s with DNA near the start point of transcription, influence promoter strength and play important regulatory roles (Haugen et al, 2006;Ross et al, 1993;Wilson and Dombroski, 1997;Zenkin et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Dissection of the Bacteriophage T4 Late Promoter Complex

Nechaev

Geiduschek

2008

Activated transcription of the bacteriophage T4 late genes is generated by a mechanism that stands apart from the common modalities of transcriptional regulation: the activator gp45 is the viral replisome's sliding clamp; two sliding clamp-binding proteins, gp33 and gp55, replace the host RNA polymerase (RNAP) σ subunit. We have mutagenized, reconfigured and selectively disrupted individual interactions of the sliding clamp with gp33 and gp55 and have monitored effects on transcription. The C-terminal sliding clamp-binding epitopes of gp33 and gp55 are perfectly interchangeable, but the functions of these two RNAP-sliding clamp connections differ, with only the gp33-gp45 linkage essential for activation. Formation of transcription-ready promoter complexes by the sliding clamp-activated wild-type T4 RNAP resists competition by high concentrations of the polyanion heparin. This avid formation of promoter complexes requires both linkages of the T4 late RNAP to the sliding clamp. Preopening the promoter compensates for loss of the gp55-gp45 but not the gp33-gp45 linkage. We interpret these findings in relation to the common model of transcriptional initiation in bacteria and highlight the roles of individual interactions of the promoter complex.

“…Whether or not ppGpp actually decreases transcription initiation depends on a promoter's intrinsic kinetic properties: only those promoters that make short-lived competitor-resistant complexes with RNAP are subject to inhibition (see Introduction and ref. 14 ). Since several of the substitutions for C-1 and C-2 increased the absolute lifetime of the rrnB P1 promoter complex (Table 3), 14 the effect of ppGpp on transcription initiation from the mutant promoters was not tested directly.…”

Section: The Intp and Ppgpp Do Not Compete For Binding To Rnap Duringmentioning

confidence: 99%

Still Looking for the Magic Spot: The Crystallographically Defined Binding Site for ppGpp on RNA Polymerase Is Unlikely to Be Responsible for rRNA Transcription Regulation

Vrentas

Gaál

Berkmen

et al. 2008

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SummaryIdentification of the RNA polymerase (RNAP) binding site for ppGpp, a central regulator of bacterial transcription, is crucial for understanding its mechanism of action. A recent high resolution x-ray structure defined a ppGpp binding site on T. thermophilus RNAP. We report here effects of ppGpp on ten mutant E. coli RNAPs with substitutions for the analogous residues within 3-4Å of the ppGpp binding site in the T. thermophilus cocrystal. None of the substitutions in E. coli RNAP significantly weakened its responses to ppGpp. This result differs from the originally-reported finding of a substitution in E. coli RNAP eliminating ppGpp function. The E. coli RNAPs used in that study likely lacked stoichiometric amounts of ω, an RNAP subunit required for responses of RNAP to ppGpp, in part explaining the discrepancy. Furthermore, we found that ppGpp did not inhibit transcription initiation by T. thermophilus RNAP in vitro or shorten the lifetimes of promoter complexes containing T. thermophilus RNAP, in contrast to the conclusion in the original report. Our results suggest that the ppGpp binding pocket identified in the cocrystal is not the one responsible for regulation of E. coli rRNA transcription initiation and highlight the importance of inclusion of ω in bacterial RNAP preparations.