Transcription control at the melting step is not yet understood. Here, band shift, cross-linking, and transcription experiments on diverse DNA probes were used with two bacterial RNA polymerase holoenzymes that differ in how they regulate melting. Data indicated that both 54 and 70 holoenzymes assume a default closed form that cannot establish single-strand binding. Upon activation the enzymes are converted to an open form that can bind simultaneously to the upstream fork junction and to the melted transcription start site. The key difference is that 54 imposes tighter regulation by creating a complex molecular switch at −12/−11; the current data show that this switch can be thrown by activator. In this case an ATP-bound enhancer protein causes 54 to alter its cross-linking pattern near −11 and also causes a reorganization of holoenzyme: DNA interactions, detected by electrophoretic mobility-shift assay. At a temperature-dependent 70 promoter, elevated temperature alone can assist in triggering conformational changes that enhance the engagement of single-strand DNA. Thus, the two factors modify the same intrinsic opening pathway to create quite different mechanisms of transcriptional regulation.
23 amino acid substitutions were made in the C7 and C3 regions of pspF⌬HTH, a protein required to convert 54 closed promoter complexes to open complexes. These mutants were assayed for transcriptional competence, for the ability to hydrolyze ATP, for their multimerization state, and for their ability to interact with 54 and its holoenzyme. C7 region mutants caused the protein to assume a compact form. This property could be mimicked by the addition of ATP, implying that compaction via C7 and ATP is part of the activation process. A number of C3 mutants were important for energy coupling, as indicated previously for several members of this activator family (1, 2). However, a patch within C3 influenced oligomerization. The C3 region was especially important in interacting with 54 during the transition state but not important in inducing 54 holoenzyme to engage the nontemplate strand of the promoter. It is proposed that both regions contain deterrent functions that prevent premature activation. Overall, the results imply unexpected roles for the C7 and C3 regions of this protein family during promoter activation.
The rate of transcription of Escherichia coli ribosomal RNA promoters is central to adjusting the cellular growth rate to nutritional conditions. The ؉1 initiating nucleotide and ppGpp are regulatory effectors of these promoters. The data herein show that in vitro transcription is also regulated by the ؉2 nucleotide. Both the ؉1 and ؉2 nucleotides act by driving polymerase into an altered conformation rather than by increasing the lifetime of transcription complexes. The unique design of the ribosomal promoters may stabilize a distorted state of polymerase that is relieved by the binding of the two nucleotides required for transcription initiation.Transcription of ribosomal RNA is the limiting step in ribosome production (1). The ribosomal promoters sense the overall availability of nutrients and respond by producing amounts of rRNA that can support the maximal growth possible given the nutritional environment. Nucleotides have been shown to be critical effectors of this promoter response. The signal nucleotide ppGpp is produced in inverse concentration to growth rate, and this may help keep the rate of rRNA transcription at an appropriate level (2, 3). The ribosomal promoters are also unusually sensitive to the concentration of the initiating nucleotide (4, 5). During transitions to slow growth, the nucleotide concentration can be depressed, and this prevents unnecessary transcription of ribosomal components (3).The mechanism by which nucleotides affect transcription from ribosomal promoters has received a great deal of attention. In the case of the initiating nucleotide, it was proposed that it works by stabilizing polymerase-promoter DNA complexes (4). This effect is thought to be specific to the ribosomal promoters because their complexes have short half-lives and are therefore uniquely unstable. ppGpp also reduces the complex half-life (6) and competes with the initiating nucleotide (5).There are uncertainties associated with this mechanism of nucleotide control of ribosomal transcription. In order for the short lifetime of ribosomal complexes to reduce transcription, dissociation would need to occur before the first RNA bond is formed, but this is typically quite rapid (7-10). How these unstable complexes are uniquely stabilized by the ϩ1 nucleotide is not known. In addition, recent experiments in other transcription systems have shown that the ϩ2 nucleotide can also have stimulatory effects. This occurs during initiation by the viral T7 RNA polymerase (11) and during elongation by the Escherichia coli RNA polymerase (12). Nucleotides that do not match the ϩ1 position on the template can stimulate the isomerization of E. coli RNA polymerase in complexes with fork-junction DNA.1 The structure of such complexes is known (13) and gives no obvious clue as to how this stabilization by nucleotides could occur.For these reasons we have re-evaluated the role of nucleotides as effectors of ribosomal transcription. The new data show that both the ϩ1 and ϩ2 nucleotides can act as effectors, unifying the properties of...
Pfs expression is required for several metabolic pathways and limits the production of autoinducer-2, a molecule proposed to play a central role in interspecies quorum sensing. The present study reveals physiological conditions and promoter DNA elements that regulate Escherichia coli pfs transcription. Pfs transcription is shown to rely on both sigma 70 and sigma 38 (rpoS), and the latter is subject to induction that increases pfs expression. Transcription is maximal as the cells approach stationary phase, and this level can be increased by salt stress through induction of sigma 38-dependent expression. The pfs promoter is shown to contain both positive and negative elements, which can be used by both forms of RNA polymerase. The negative element is contained within the overlapping dgt promoter, which is involved in purine metabolism. Consideration of the physiological roles of sigma 38 and dgt leads to a model for how autoinducer production is controlled under changing physiological conditions.The pfs gene, encoding the 5Ј-methylthioadenosine/S-adenosylhomocysteine nucleosidase enzyme, is required for polyamine synthesis, adenine and methionine salvage, removal of toxic metabolites, and creation of luxS-dependent autoinducers (AIs) of quorum sensing in Escherichia coli and related bacteria (5,22,26). As quorum sensing involves cell-to-cell signaling, it would be expected to be a highly regulated process. Gene fusions have shown that the pfs-luxS pathway of AI-2 synthesis is limited by the expression of pfs rather than luxS (3). Pfs expression rises as cells approach stationary phase and then falls. AI-2 synthesis follows a similar pattern (31). AI-2 is excreted until stationary phase is reached, when it begins to be imported (27,31). The sources of these regulatory events are not known, and their significance with respect to quorum sensing is not well understood. In interspecies quorum sensing, it has been proposed that products of the pfs/luxS pathway, AI-2 molecules, accumulate from multiple bacterial species and then signal the population to behave coordinately (12, 32). The issue is important because quorum sensing is a widespread phenomenon and has been invoked as a potential target of therapeutic intervention (2).Because the expression of pfs rises as stationary phase approaches (3), we considered the possibility that the source of the rise might be induction of rpoS-dependent transcription. rpoS encodes sigma 38, which is induced in response to multiple stresses and directs the transcription of numerous genes that deal with physiological stress (10). Its initial expression pattern mimics that seen for pfs; sigma 38 is induced as cells sense the stresses that will lead to the halt in growth that is characteristic of stationary phase (14). If sigma 38 were the source of pfs regulation, this would have implications for events such as quorum sensing, which is proposed to be controlled by the AI system.We now report that rpoS-dependent expression is indeed the source of pfs induction. Overall, pfs transcripti...
During promoter engagement, RNA polymerase must change conformation or isomerize to its active form. These data show that high concentrations of nucleotides assist this isomerization. When binding to fork junction DNA probes is monitored, isomerization can occur without the need for the DNA that overlaps the transcription start site. When the start site is present, nucleoside triphosphates cause polymerase to change conformation in a way that drives cross-linking to the +1 position on the template strand. Preincubation of transcription complexes with 2 mM initiating nucleotide can drive formation of heparin-resistant complexes under conditions in which isomerization is limiting. It is proposed that complete polymerase isomerization can require nucleotide binding, which can assist formation of the active site that engages the transcription start site.
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