Guanosine 3′,5′‐bis(diphosphate) (ppGpp)‐Dependent Inhibition of Transcription from Stringently Controlled Escherichia Coli Promoters can be Explained by an Altered Initiation Pathway that Traps RNA Polymerase

Abstract

An in vitro analysis was performed to investigate the inhibitory mechanism of the global regulatory substances guanosine 3'$-bis(diphosphate) (ppGpp) and guanosine 3'-diphosphate 5'-triphosphate (pppGpp) during initiation of transcription. Three promoters with well known differential ppGpp sensitivities in vivn were studied: the Escherichiu cnli rrnB P2 promoter that is only weakly ppGpp dependent; a P2 base change variant (P2F) that confers both stringent and growth rate regulation; and the completely unregulated PtacI promoter. The in vivo ppGpp dependency for all three promoters was verified in vitro in multiple round transcription reactions, reflecting a combination of the effects at initiation, promoter clearance, and elongation. In the main part of our study, we concentrated on the contribution of initiation complex formation to the overall inhibition of transcription. Kinetic measurements of complex association and dissociation revealed that at sensitive promoters (p)ppGpp triggered an alternative initiation pathway by RNA polymerase. This involved the stabilization of the initial closed complexes, and impeded open complex formation. Subsequently formed ternary complexes were structurally altered. Based on the above findings, we propose a model which suggests that ppGpp-altered RNA polymerases are preferentially bound and enter the alternative pathway. Thus, discrimination is obtained at early steps of initiation, which causes efficient inhibition at later steps of the transcription cycle probably involving promoter clearance and elongation.Keywords: Escherichia coli RNA polymerase ; guanosine 3',5'-bis(diphosphate) ; ribosomal RNA promoter; transcription regulation; stringent control.The two major regulatory systems controlling ribosomal RNA synthesis in bacteria are stringent control and growth rate regulation. Both mechanisms are related to changes in the level of the effector nucleotides (p)ppGpp. Transcription of all seven E. coli rRNA operons is directed by two tandem promoters, P1 and P2. Upstream promoters P1 are strongly regulated by enhanced guanosine 3',5'-bis(diphosphate) (ppGpp) levels, while downstream promoters P2, although regulated under certain circumstances [I], show a less clear ppGpp sensitivity [2-41. However, according to recent in vivo studies transcription from P2 is also repressed under conditions of stringent control [5-81. All ribosomal RNA PI promoters, but not P2 promoters, contain the GCGC discriminator motif important for stringency and growth rate regulation. P2 promoters have either GCAC ( r r A , B, C, G) or ACGC (rrnD, E, H) sequences at the same position. Recently, the promoter P2 variant P2F was constructed by a single base transition from A to G at position -5 [9], which generates a perfect discriminator. The P2F promoter has been shown in vivo to be stringently and growth rate regulated, exactly as PI, whereas the wild-type P2 was almost insensitive under the same conditions [9]. The molecular mechanism by which ppGpp inhibits transcription is completely unknown.We...

Control of rRNA Synthesis in Escherichia coli : a Systems Biology Approach

“…It might appear that such a proof can only come from in vitro experiments. Numerous research groups have shown in vitro inhibition of P1 activity by ppGpp (10,50,53,65,66,98). However, depending on the assay conditions (including the degree of superhelicity of the templates), the in vitro results have differed quantitatively from in vivo observations, presumably because complex in vivo conditions cannot yet be reproduced in vitro.…”

“…These and further observations suggested that ppGpp is involved in the control of rRNA synthesis. Subsequently, it was observed that ppGpp specifically inhibits rRNA synthesis in vitro (50, 65, 66 130-132), presumably by reducing the affinity of the RNA polymerase to stable RNA promoters (50,53,65,66,98). Thus, unless these in vitro effects are artifactual, they indicate that ppGpp is a direct effector and responsible for at least part of the in vivo inhibition of rRNA synthesis during the stringent response.…”

In Vivo and In Vitro Effects of Integration Host Factor at the DmpR-Regulated ς 54 -Dependent Po Promoter

“…Reactions were performed under the same buffer conditions and protein concentrations used in the in vitro tran- scription assays. It should be noted that open complexes could not be detected using this linear template without the stabilizing effect of the addition of the two initiating nucleotides (GTP and CTP), as has been observed with unstable E 70 open complexes (18,20). Since dATP used as the regulator nucleotide for hydrolysis (see Materials and Methods) can be incorporated inefficiently into transcripts, a nascent transcript of up to 14 nucleotides could be formed (see Fig.…”

Differential stringent control of Escherichia coli rRNA promoters: effects of ppGpp, DksA and the initiating nucleotides

“…It is also known that binary open complexes between RNA polymerase and promoters negatively regulated by ppGpp are intrinsically unstable. Such promoters can be stabilized by their corresponding iNTPs, highlighting the importance of the starting nucleotide in the negative regulation of ppGpp-dependent promoters (Barker et al, 2001;Gourse, 1988;Heinemann & Wagner, 1997;Langert et al, 1991). To assess the involvement of the iNTPs in the weaker ppGpp sensitivity of the rrnD P1 promoter in vivo demonstrated above (Fig.…”

“…The diagrams below show the percentage residual promoter activity from two experiments after serine hydroxamate addition, normalized to the RNA 1 intensity. Gourse, 1988;Heinemann & Wagner, 1997;Langert et al, 1991). To assess the involvement of the iNTPs in the weaker ppGpp sensitivity of the rrnD P1 promoter in vivo demonstrated above ( Fig.…”