Different roles for basic and aromatic amino acids in conserved region 2 of Escherichia coli sigma(70) in the nucleation and maintenance of the single-stranded DNA bubble in open RNA polymerase-promoter complexes.

Amino acid residues in region 2 of final sigma(70) have been shown to play an important role in the strand separation step that is necessary for formation of the functional or open RNA polymerase-promoter complex. Here we present a comparison of the roles of basic and aromatic amino acids in the accomplishment of this process, using RNA polymerase bearing alanine substitutions for both types of amino acids in region 2. We determined the effects of the substitutions on the kinetics of open complex formation, as well as on the ability of the RNA polymerase to form complexes with single-stranded DNA, and with forked DNA duplexes carrying a single-stranded overhang consisting of bases in the -10 region. We concluded that two basic amino acids (Lys(414) and Lys(418)) are important for promoter binding and demonstrated distinct roles, at a subsequent step, for two aromatic amino acids (Tyr(430) and Trp(433)). It is likely that these four amino acids, which are close to each other in the structure of final sigma(70), together are involved in the nucleation of the strand separation process.

Green fluorescent protein as a quantitative reporter of relative promoter activity in E. coli.

Green fluorescent protein (GFP) has become a valuable tool for the detection of gene expression in prokaryotes and eukaryotes. To evaluate its potential for quantitation of relative promoter activity in E. coli, we have compared GFP with the commonly used reporter gene lacZ, encoding beta-galactosidase. We cloned a series of previously characterized synthetic E. coli promoters into GFP and beta-galactosidase reporter vectors. Qualitative and quantitative assessments of these constructs show that (a) both reporters display similar sensitivities in cells grown on solid or liquid media and (b) GFP is especially well suited for quantitation of promoter activity in cells grown on agar. Thus, GFP provides a simple, rapid and sensitive tool for measuring relative promoter activity in intact E. coli cells.

Promoter interference in a bacteriophage lambda control region: effects of a range of interpromoter distances.

The p(R) and p(RM) promoters of bacteriophage lambda direct transcription in divergent directions from start sites separated by 83 phosphodiester bonds. We had previously shown that the presence of an RNA polymerase at p(R) interfered with open complex formation at p(RM) and that this effect was alleviated by the deletion of 10 bp between the two promoters. Here we present a detailed characterization of the dependence of the interference on the interpromoter distance. It was found that the reduced interference between the two promoters is unique to the 10-bp deletion. The relief of interference was demonstrated to be due to the facilitation of a step subsequent to RNA polymerase binding to the p(RM) promoter. A model to explain these observations is proposed. A search of known Escherichia coli promoters identified three pairs of divergent promoters with similar separations to those investigated here.

Equilibrium and kinetic parameters of the sequence-specific interaction of Escherichia coli RNA polymerase with nontemplate strand oligodeoxyribonucleotides.

The specific recognition by Escherichia coli RNA polymerase of single-stranded oligodeoxyribonucleotides (oligos) with the sequence of the -10 promoter region on the nontemplate strand has been studied. Binding was monitored by observing the increase in fluorescence of 2-aminopurine residues incorporated in the oligos. The effects of salt on the rates of formation and dissociation of RNA polymerase.oligo complexes are relatively small, from which we conclude that electrostatic interactions contribute minimally to the favorable binding free energy. From the convex temperature dependence of ln Ka (Ka is the equilibrium association constant), we infer that a large apparent negative heat capacity, of 1-2 kcal M-1 K-1, accompanies complex formation, which is interpreted as due to a conformational change in RNA polymerase. Contrary to expectation, the forward rate constant for binding of oligos is more than 10-fold smaller than that for open complex formation at strong promoters. This suggests that in comparison to an oligo, promoter DNA may be better able to accelerate this required conformational change in the RNA polymerase. Oligo binding was shown to compete with the interaction between RNA polymerase and promoters, indicating that the two bind to overlapping sites on the RNA polymerase

Promoter recognition by Escherichia coli RNA polymerase: effects of the UP element on open complex formation and promoter clearance.

Escherichia coli promoters for transcription of ribosomal and tRNAs are greatly activated by an A+T-rich "UP" element upstream of the -35 region. These same promoters have also been found to otherwise deviate in several respects from the consensus promoter sequence. Here we present the results of a kinetic characterization of the interaction of Escherichia coli RNA polymerase with UP element-containing promoters which by virtue of consensus or near-consensus sequence features should be among the most optimal that can be encountered by Escherichia coli RNA polymerase. We show that for such promoters, (1) the second-order rate constant describing formation of the initial (closed) complex is close to that expected for a diffusion-limited process, (2) the extent of activation by the UP element is temperature-sensitive, (3) the UP element accelerates a process after DNA binding by RNA polymerase, and (4) the presence of the UP element delays promoter clearance upon addition of nucleoside triphosphates to preformed RNA polymerase-promoter complexes. Finally, we provide evidence in support of models which describe the DNA melting process accompanying open complex formation as initiating in the -10 promoter region and progressing in the downstream direction.

Spectroscopic determination of open complex formation at promoters for Escherichia coli RNA polymerase.

A considerable amount of effort has been expended studying the kinetics of association of Escherichia coli RNA polymerase with promoter DNA in forming the open complex. Strand separation occurs over about 12 base pairs and includes the transcription start site. However, these efforts have been significantly hampered by the lack of a sensitive, real time method by which formation of an open complex could be assayed. Here, we employ short (86 bp) synthetic promoters with 2-aminopurine (2-AP) substitutions in the region that becomes single-stranded to spectroscopically monitor open complex formation. We demonstrate that promoters bearing the substitutions behave in a manner similar to that of those containing only the four common bases with respect to both the region of strand separation and start site selection. Open complex formation was found to yield an increased fluorescence signal with an emission maximum characteristic of 2-aminopurine. This spectroscopic assay for open complex formation was found to be well-suited to the investigation of a strong promoter, allowing open complex formation to be followed over a time scale of seconds with a stopped flow apparatus. The introduction of two additional nonconsensus base pairs in the -35 region resulted in a promoter for which open complex formation was 100-fold slower. The same substrates were also used to monitor the promoter re-annealing that ensues upon initiation of RNA synthesis. Similar rates for this process were observed for the two promoter variants employed in this study.

Upstream interactions at the lambda pRM promoter are sequence nonspecific and activate the promoter to a lesser extent than an introduced UP element of an rRNA promoter.

The rightward regulatory region of bacteriophage lambda contains two promoters, pRM and pR, which direct the synthesis of nonoverlapping divergent transcripts from start sites 82 bp apart. Each of the two promoters has an upstream (A+T)-rich region (ATR) within the sequence from -40 to -60 where in the rrnB P1 promoter a stretch of 20 (A+T) bp greatly stimulates promoter function. Here we present an investigation of the possible functional significance of pRM's ATR. We determined the effects on RNA polymerase-pRM promoter interaction both of (G+C) substitutions in the ATR and of amino acid substitutions in the alpha subunit, known to affect the upstream interaction. We find small (two- to threefold) effects of selected mutations in the alpha subunit on open complex formation at pRM. However, the (presumably upstream) interactions underlying these effects are sequence nonspecific, as they are not affected by (G+C) substitutions in the ATR. Substitution of the 20-bp UP element of the rrnB P1 promoter between positions -40 and -60 at pRM stimulates open complex formation to a considerably greater extent (5- to 10-fold). Results from kinetic studies indicate that on this construct the UP element mainly accelerates a step subsequent to the binding of RNA polymerase, although it may also facilitate the binding event itself. Less extensive studies likewise provide evidence for a two- to threefold activation of pR by upstream interactions. The possible involvement of the alpha subunit in the previously characterized (e.g., B. C. Mita, Y. Tang, and P. L. deHaseth, J. Biol. Chem. 270:30428-30433, 1995) interference of pR-bound RNA polymerase with open complex formation at pRM is discussed.

Interference of PR-bound RNA polymerase with open complex formation at PRM is relieved by a 10-base pair deletion between the two promoters.

Bacteriophage lambda promoters PR and PRM direct RNA synthesis in divergent orientations from start sites 82 base pairs apart. We had previously determined that the presence on the same DNA fragment of a wild type PR promoter interfered with the utilization of the PRM promoter. The results reported here concern the effects of changing the distance between the start sites by insertion or deletion of 5 or 10 base pairs. Three different techniques (run-off transcription, gel mobility shift, and permanganate probing) were employed to monitor complex formation at PRM. Unexpectedly we find that deletion of 10 base pairs between the start sites abolishes the interference, whereas insertion of 10 base pairs does not. Deletion of 5 base pairs, however, essentially prevents joint complex formation at PR and PRM. These findings suggest several ways in which for the wild type separation of the two promoters the utilization of PRM could be affected by an RNA polymerase at PR. In addition to direct steric interference, these include the obstruction of access to DNA sites necessary for optimal contact with the RNA polymerase.

Open complex formation by Escherichia coli RNA polymerase: the mechanism of polymerase-induced strand separation of double helical DNA.

Escherichia coli RNA polymerase is able to site-specifically melt 12 bp of promoter DNA at temperatures far below those normally associated with DNA melting. Here we consider several models to explain how RNA polymerase destabilizes duplex DNA. One popular model proposes that upon binding to the promoter, RNA polymerase untwists the spacer DNA between the -10 and -35 regions, which results in a destabilization of the -10 region at a TA base step where melting initiates. Promoter untwisting may result, in part, from extensive wrapping of the DNA around RNA polymerase. Formation of the strand-separated open complex appears to be facilitated by specific protein-DNA interactions which occur predominantly on the non-template strand. Recent evidence suggests that these include important contacts with sigma factor region 2.3, which we propose binds the displaced single strand of DNA.

Mutations in sigma factor that affect the temperature dependence of transcription from a promoter, but not from a mismatch bubble in double-stranded DNA.

Specificity of promoter utilization in bacterial RNA polymerases is imparted by a class of proteins referred to as sigma factors. Conserved region 2.3 of these proteins is thought to play a role in the strand separation process that occurs during the formation of an initiation-competent RNA polymerase-promoter complex. We have used a heterologous system consisting of Escherichia coli core RNA polymerase and Bacillus subtilis sigma A to probe the effects of amino acid substitutions in region 2.3. In agreement with previous work [Juang & Helmann (1994) J. Mol. Biol. 235, 1470-1488] we observe that several amino acid substitutions exacerbate the deleterious effect of low temperature on promoter-dependent initiation. On the other hand, no such enhanced cold sensitivity is found with double-stranded templates that contain short "bubbles" of single-stranded DNA, indicating that the DNA-melting defect imposed by these mutant sigma factors can be suppressed by the use of such bubble templates. These results support the involvement of region 2.3 in the strand separation process that accompanies open complex formation at promoters.

A mismatch bubble in double-stranded DNA suffices to direct precise transcription initiation by Escherichia coli RNA polymerase.

Formation of a transcription-competent "open" complex between Escherichia coli RNA polymerase and a promoter, where base pairing is disrupted over a region of 12 base pairs including the start site of transcription, is a complex process involving at least three steps: recognition of specific DNA sequences, a conformational change in RNA polymerase, and DNA melting. By using synthetic constructs devoid of promoter-specific sequences, we show here that a mismatch bubble of 12 base pairs suffices to direct transcription initiation in divergent directions from its edges, reflecting the absence of polarity determinants for RNA polymerase binding. Bubble transcription is obtained with both core polymerase and holoenzyme, but efficient formation of heparin-resistant initiation complexes requires the sigma (specificity) factor. Based on these results it is likely that the sigma factor blocks access of the heparin to a site on the holoenzyme.

A new start site for Escherichia coli RNA polymerase at an engineered short region of non-complementarity in double-stranded DNA.

We have constructed versions of the bacteriophage PRM promoter containing short (9 or 12 base pairs) regions of DNA mismatches ("bubble") which include the authentic transcription start site of the unmodified promoter. These constructs direct transcription initiation at positions near the genuine PRM start site. In addition a new start site (designated Pbub) is observed in the region of non-complementarity, from which RNA synthesis proceeds in the opposite direction. The ability to initiate the divergent transcripts is specific to holo enzyme. Mapping of the Pbub start sites shows that they are but a few base pairs upstream of the edge of the bubble. Thus, with respect to the single-stranded region, the location of the start site is no different for Pbub than it is for open complexes at promoters. Compared with an unmodified PRM promoter, the region protected by RNA polymerase from digestion by DNase I is extended in the downstream direction (with respect to the PRM start) at the promoters bearing mismatches; this is consistent with the binding of the divergently transcribing RNA polymerase. Interestingly, cI protein represses rather than activates RNA synthesis originating in the PRM direction, indicating yet another aspect in which the complexes formed at these constructs differ from open complexes at the unmodified promoter.

Promoter recognition by Escherichia coli RNA polymerase. Effects of single base pair deletions and insertions in the spacer DNA separating the -10 and -35 regions are dependent on spacer DNA sequence.

Escherichia coli RNA polymerase contacts promoter DNA at two upstream regions separated by a spacer DNA. We had previously studied the effects of substitutions of simple DNA sequences in a stretch of the spacer DNA devoid of any known specific contacts with RNA polymerase. It was found that substitution of nine consecutive nonalternating dG-dC base pairs, but not nine alternating dG-dC base pairs, impaired promoter function. We proposed that this effect was due to the fact that the oligo(dG)-oligo(dC) sequence adopted a conformation (possibly A-helical) resulting in a reduction in its length and twist as compared with the B-form DNA of the alternating sequence. Here we test this hypothesis by combining the substitutions with single base pair insertions and deletions in the spacer DNA, which affect the length and the twist in known ways. Deletion and substitutions equally affect the activities of promoters with the presumed B-DNA substitutions. However, for promoters bearing the oligo(dG)-oligo(dC) substitution, a deletion in the spacer DNA impairs promoter activity to a much greater extent than the insertion of a base pair. This asymmetry is consistent with our hypothesis that the deleterious effects of the substitution are due to its having the reduced twist and/or length characteristic of A-DNA. Additionally, we present data that concern the sequence requirements for adoption of this structure that leads to reduced promoter function.

Interference by PR-bound RNA polymerase with PRM function in vitro. Modulation by the bacteriophage lambda cI protein.

Activation of the weak PRM promoter by cI protein is an essential process in the establishment of lysogeny. Much evidence has accumulated that cI protein binds cooperatively to the operators OR1 and OR2 and that protein at the OR2 site contacts RNA polymerase to facilitate open complex formation at the PRM promoter. We had shown previously in vitro that RNA polymerase situated at the nearby PR promoter could interfere with open complex formation at PRM and that an additional mechanism of PRM activation in vitro involved cI-mediated RNA polymerase exclusion from PR. Here we further characterize this second indirect mode of activation. We demonstrate the addition of cI and inactivation of the PR promoter activate open complex formation at PRM similarly over the temperature range from 37 to 20 degrees C in which the extent of activation decreases from 8- to 2-fold. We also show that the binding of cI protein to OR1 is sufficient to effect an increase in the rate of synthesis of abortive RNA products at PRM. This result is difficult to explain based on direct cI-RNA polymerase contacts alone but is readily interpreted in terms of our previously proposed model involving the exclusion of an interfering RNA polymerase from binding at PR.

RNA polymerase bound to the PR promoter of bacteriophage lambda inhibits open complex formation at the divergently transcribed PRM promoter. Implications for an indirect mechanism of transcriptional activation by lambda repressor.

We demonstrate that RNA polymerase bound at the PR promoter of bacteriophage lambda can repress transcription initiation from the divergently transcribed PRM promoter in vitro. Using abortive initiation and run-off transcription experiments we show that inactivating mutations introduced into either the -10 or -35 regions of PR result in a significant increase in the rate of formation of transcriptionally competent complexes at the PRM promoter. This is due primarily to an increase in the rate constant for the isomerization of closed to open complexes. Gel shift and DNase I footprinting experiments were employed to further define the mechanism by which PR sequences mediate PRM repression. From these assays we were able to conclude that the formation of an open complex at the PR promoter did not exclude RNA polymerase from binding at PRM. Rather, initiation at PRM was impaired because closed complexes must isomerize in the presence of an open complex already situated at the PR promoter. Extensive evidence has been obtained previously indicating that lambda repressor activates transcription directly by contacting RNA polymerase situated at the PRM promoter. Results presented here raise the possibility that an additional mechanism could be operative, whereby lambda repressor indirectly activates PRM transcription by excluding RNA polymerase from the PR promoter.

Bacteriophage lambda promoters pL and pR: sequence determinants of in vivo activity and of sensitivity to the DNA gyrase inhibitor, coumermycin.

Sequence encompassing the region between bp -43 and +8 of the pL and pR promoters of bacteriophage lambda, as well as sequence variants of these promoters, were compared with respect to their ability to drive a promoterless cat gene in vivo. For both pL- and pR-based promoters, variants with one nonconsensus bp rather than the consensus promoter were found to be maximally active. Determination of promoter function in CSH26 and C600 revealed a marked strain dependence in the activity of some promoter variants. In response to the antibiotic coumermycin, which effects a reduction in DNA superhelical density in vivo, promoters were found to be activated, inhibited or unaffected, depending on their sequence. No simple correlation between a particular response and sequence features of a promoter has become apparent.

Saturation mutagenesis of an Escherichia coli rRNA promoter and initial characterization of promoter variants.

Using oligonucleotide synthesis techniques, we generated Escherichia coli rrnB P1 (rrnB1p according to the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoter fragments containing single base substitutions, insertions, deletions, and multiple mutations, covering the whole length of the promoter including the upstream activation sequence (UAS). The activities of 112 mutant promoters were assayed as operon fusions to lacZ in lambda lysogens. The activities of most mutants with changes in the core promoter recognition region (i.e., substitutions, insertions, or deletions in the region of the promoter spanning the -10 and -35 E. coli consensus hexamers) correlated with changes toward or away from the consensus in the hexamer sequences or in the spacing between them. However, changes at some positions in the core promoter region not normally associated with transcriptional activity in other systems also had significant effects on rrnB P1. Since rRNA promoter activity varies with cellular growth rate, changes in activity can be the result of changes in promoter strength or of alterations in the regulation of the promoter. The accompanying paper (R. R. Dickson, T. Gaal, H. A. deBoer, P. L. deHaseth, and R. L. Gourse, J. Bacteriol. 171:4862-4870, 1989) distinguishes between these two alternatives. Several mutations in the UAS resulted in two- to fivefold reductions in activity. However, two mutants with changes just upstream of the -35 hexamer in constructs containing the UAS had activities 20- to 100-fold lower than the wild-type level. This collection of mutant rRNA promoters should serve as an important resource in the characterization of the mechanisms responsible for upstream activation and growth rate-dependent regulation of rRNA transcription.

Identification of promoter mutants defective in growth-rate-dependent regulation of rRNA transcription in Escherichia coli.

We measured the activities of 50 operon fusions from a collection of mutant and wild-type rrnB P1 (rrnB1p in the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoters under different nutritional conditions in order to analyze the DNA sequence determinants of growth rate-dependent regulation of rRNA transcription in Escherichia coli. Mutants which deviated from the wild-type -10 or -35 hexamers or from the wild-type 16-base-pair spacer length between the hexamers were unregulated, regardless of whether the mutations brought the promoters closer to the E. coli promoter consensus sequence and increased activity or whether the changes took the promoters further away from the consensus and reduced activity. These data suggest that rRNA promoters have evolved to maintain their regulatory abilities rather than to maximize promoter strength. Some double substitutions outside the consensus hexamers were almost completely unregulated, while single substitutions at several positions outside the -10 and -35 consensus hexamers exerted smaller but significant effects on regulation. These studies suggest roles for specific promoter sequences and/or structures in interactions with regulatory molecules and suggest experimental tests for models of rRNA regulation.