Mutations at position -10 in the lambda PR promoter primarily affect conversion of the initial closed complex (RPc) to a stable, closed intermediate (RPi).

The effects of mutations of --10 T:A to A:T, C:G, or G:C in the lambda P(R) promoter on formation of transcriptionally competent open complexes were studied by DNAse I footprinting, KMnO(4)-sensitivity, and abortive initiation kinetic analysis. The mutations --10A (T:A --> A:T) and --10C significantly reduce k(f), the composite rate constant for conversion of closed complexes (RP(c)) to open complexes (RP(o)) but do not affect K(B), the equilibrium constant for formation of closed complexes. Unlike the other mutants or wild-type P(R), the mutation with the largest effect on open complex formation, --10G (T:A --> G:C), substantially decreases the occupancy of the promoter. When reduced occupancy is taken into account, the calculated effect of the mutation on k(f) is a 20-fold reduction. Analysis of open complex formation by a three-step pathway that includes an additional intermediate, RP(i), indicates that the primary effect of all three mutations is a reduction in the rate of isomerization of RP(c) to RP(i), which precedes DNA strand separation. Thus, RNA polymerase holoenzyme must recognize specific base pairs in the --10 region of P(R) while the DNA is still double-stranded. Comparison of the observed level of stable complexes (RP(i) plus RP(o)) with the level of productive complexes (RP(o)) indicates that the --10G mutation may also affect the equilibrium between RP(i) and RP(o) at 37 degrees. Open complexes formed at the three mutant promoters are approximately 3-5 times less stable at 37 degrees than those formed at wild-type P(R).

Effects of mutations in the Pseudomonas putida miaA gene: regulation of the trpE and trpGDC operons in P. putida by attenuation.

Tn5 insertion mutants defective in regulation of the Pseudomonas putida trpE and trpGDC operons by tryptophan were found to contain insertions in the P. putida miaA gene, whose product (in Escherichia coli) modifies tRNA(Trp) and is required for attenuation. Nucleotide sequences upstream of trpE and trpG encode putative leader peptides similar in sequence to leader peptides found in other bacterial species, and the phenotypes of the mutants strongly suggest that transcription of these operons is regulated solely by attenuation.

Changes in the 17 bp spacer in the P(R) promoter of bacteriophage lambda affect steps in open complex formation that precede DNA strand separation.

Tau plots and temperature-shift experiments were used to determine which step in the formation of transcriptionally-competent open complexes is affected by changing the length of the 17 bp spacer separating the -10 and -35 consensus regions of the P(R) promoter of bacteriophage lambda. Abortive initiation assays at 37 degrees C indicate that the primary effect of insertion of a base-pair, thereby increasing spacer length to 18 bp, is a decrease in k(f), the rate constant for conversion from closed (RP(c)) to open (RP(o)) complexes, by approximately a factor of 4. The mutation did not significantly affect K(B), the equilibrium constant for formation of closed complexes, and decreased K(B)k(f) by a factor of 3. Deletion of a bp to create a 16 bp spacer had a much greater effect, decreasing the measured value of k(f) by a factor of about 25 to 30, and K(B)k(f) by a factor of 7 to 8. When the values of the parameters for the deletion mutant were corrected for incomplete occupancy of RP(o) at equilibrium, the effects of the deletion were even greater. In particular, the corrected value of K(B)k(f) was about 15 times lower than the corresponding value for two promoters with wild-type spacing. Based on temperature shift experiments, the changes in spacer length did not affect the equilibrium at 20 degrees C between RP(i), a stable intermediate in which DNA strands are not separated, and RP(o). Although differential sensitivity of single-stranded bases to KMnO(4) indicated that in about 20% of the open complexes at 20 degrees C the DNA strands are not fully separated (RP(o1)), the distribution between these complexes and RP(o2) (DNA strands fully separated) was also not affected significantly by changes in spacer length. Thus, changes in spacer length primarily affect k(2), the rate constant for conversion of RP(c) to RP(i), which corresponds to a nucleation of DNA strand-separation. Application of published data and/or algorithms for determining effects of nucleotide sequence on twist angle or rise at individual bp steps does not provide a simple explanation of the difference in promoter strength between P(R) derivatives with 16 bp spacing and those with 18 bp spacing.

Recognition of binding sites I and II by the TrpI activator protein of pseudomonas aeruginosa: efficient binding to both sites requires InGP even when site II is replaced by site I.

TrpI protein, the activator of transcription of the trpBA operon of three species of fluorescent Pseudomonads, bends the DNA when it forms either of two well-characterized complexes with the trpBA regulatory region. In complex 1, TrpI is bound only to its strong binding site (site I), whereas in complex 2, which is required for activation of the trpBA promoter, TrpI is bound both to site I and to the weaker site II. Indoleglycerol phosphate (InGP) strongly stimulates formation of complex 2 and is required for activation. The present study focuses on the binding of TrpI to DNA containing a duplication of site I and the effect of the duplication on TrpI-induced DNA bending. We find that even on DNA containing a tandem (direct or inverted) duplication of site I, the formation of DNA-TrpI complexes with both sites occupied is strongly stimulated by InGP. Thus, even when TrpI binding to two adjacent sites needs not be cooperative, InGP significantly promotes the formation of complex 2. Gel binding data indicate that InGP can have several effects: (1) TrpI molecules bound to either of two adjacent strong binding sites appear to interfere with binding to the other site; InGP relieves this apparent interference. (2) InGP increases the intrinsic affinity of TrpI for sites I and II and/or enhances cooperative TrpI binding to adjacent DNA sites. Furthermore, a third molecule of TrpI can form a footprint adjacent to the duplication on DNA containing a direct (but not inverted) repeat of site I, indicating that TrpI bound to site I is oriented asymmetrically in spite of the quasi-symmetry of the binding site. The calculated bending angle for DNA in complex 2 is increased by approximately 20 degrees when site I is substituted in either orientation for site II; thus, on DNA containing a site I duplication, the bending angle of complex 2 is nearly twice that of complex 1.

DNA bending by the TrpI protein of Pseudomonas aeruginosa.

TrpI protein, the activator of transcription of the trpBA operon of fluorescent pseudomonads, bends the DNA when it forms either of two well-characterized complexes with the trpBA regulatory region. In complex 1, with TrpI bound only to its strong binding site (site I), the calculated bending angle is 65 to 67 degrees and the center of bending is in the middle of site I. In complex 2, which is required for activation of the trpBA promoter, with TrpI bound both to site I and to the weaker site II, the bending angle is increased to 89 to 90 degrees and the center of bending is at the site I-site II boundary. Indoleglycerol phosphate (InGP), which strongly stimulates formation of complex 2 and is required for activation, does not affect the bending angle of either complex. However, a mutation (-10C/11C) shown previously to affect activation has a small but detectable effect on bending, reducing the calculated bending angle to 83 to 86 degrees. These results suggest a way that DNA bending and InGP may be important for activation.

Direct and indirect effects of mutations in lambda PRM on open complex formation at the divergent PR promoter.

A detailed kinetic analysis demonstrates that, in vitro, mutations in the PRM promoter of bacteriophage lambda can increase the rate of open complex formation at the divergent, lytic promoter PR in either of two ways. (1) PRM- mutations, typified by PRMKM11, indirectly stimulate PR by eliminating interference from RNA polymerase (RNAP) molecules bound at wild-type PRM. This effect can be observed only when PR is itself mutated because open complexes normally form so rapidly at wild-type PR that they are unaffected by PRM. It has been shown previously that PR and PRM can be occupied simultaneously by RNAP, suggesting that interference from PRM is mediated at a step subsequent to binding of RNAP to PR. This conclusion is supported by kinetic data, which indicate that inactivating PRM affects PRx3 by increasing kf, the rate of isomerization of closed to open complexes, four- to fivefold. (2) In addition to its indirect effect, the mutation PRM116, which is located at -33 with respect to PRM and -50 with respect to PR, directly increases the intrinsic strength of PR. PRM116 increases from 11 to 12 the number of A:T or T:A base-pairs in a 12 bp AT-rich sequence located between 47 and 58 bp upstream from PR; we suggest that this upstream sequence contributes directly to PR promoter strength. We also show that the PRx3 mutation causes a 100-fold decrease in kf. This result indicates that the -35 consensus region plays a major role in the isomerization of closed to open complexes at PR.

A cryptic promoter in the O(R) region of bacteriophage lambda.

A cryptic promoter, designated P alpha, initiates transcription within the O(R) region of bacteriophage lambda. Transcription from P alpha proceeds in the direction of the cI repressor gene from sites 46 and 48 bp preceding the PRM transcription start site. P alpha is likely to compete with both PR and PRM for formation of open complexes, since it is only active when PR is mutated and can be suppressed by mutations that increase PRM activity. In addition, transcription initiation at P alpha is blocked by lambda repressor. Kinetic analysis of relative abundance of the products of in vitro transcription indicated that P alpha was approximately 1/3 as strong as PRM. However, a P alpha mutation had little effect on KBkf (the association rate constant) for PRM. These observations can be explained by the finding that open complexes formed at P alpha are relatively unstable (half-life = 20 to 25 min). Dissociation of RNA polymerase from P alpha allows additional open complexes to form at PR or PRM, and thus the apparent strength of P alpha decreases with increasing preincubation times.

Modulation of P(RM) activity by the lambda PR promoter in both the presence and absence of repressor.

When the transcription startsites of the phage lambda promoters PRM and PR are separated by 82 bp (the wild-type spacing), mutating PR increases the rate of open complex formation at PRM at all RNA polymerase (RNAP) concentrations tested in vitro. This is reflected in a fourfold increase in kappa f (the rate constant for isomerization of closed to open complexes) and a threefold decrease in KB (the equilibrium constant for formation of closed complexes). These effects of mutating PR resemble qualitatively those we observed when the separation between the two promoters was decreased by a single base-pair, but are quantitatively less dramatic. Although mutating PR has the additional effect of uncovering a weak promoter, P alpha, which overlaps both PRM and PR, the presence of P alpha does not account for the effects of PR mutations on open complex formation at PRM. In fixed-time assays at a single RNAP concentration, repressor stimulated PRM approximately threefold on a PR- template, indicating that activation is mediated substantially by a direct interaction between repressor and RNAP. That is, activation of PRM is not merely an indirect consequence of repressing PR. Kinetic data confirm this conclusion. In a PR- genetic background, repressor increased kappa f six- to eightfold and decreased KB approximately twofold. Similar results were obtained when OR3 was mutated, indicating that the effect on KB is not due to repressor binding to OR3. Thus, repressor causes a significant increase in the rate of open complex formation at PRM even when PR is inactive. On a PR+ template, 75 nM repressor stimulated PRM by increasing kappa f eightfold, with no effect on KB, which agrees with previous results. However, increased repressor concentrations stimulated kappa f by an additional factor of two to four, indicating that previous experiments underestimated the effect of repressor on kappa f. At the same time, increasing the repressor concentration decreased KB for PRM on a wild-type template. At the highest repressor concentration tested (275 nM), KB decreased 15-fold, presumably due to OR3-mediated repression of PRM. However, at an intermediate repressor concentration (170 nM) values of kappa f and KB for PRM on a PR+ template were in close agreement with the corresponding parameters obtained on a PR- template. These data lead us to suggest that repressor causes a decrease in KB for PRM on both a PR+ and a PR- template independent of its ability to bind to OR3.

Effects of a single base-pair deletion in the bacteriophage lambda PRM promoter. Repression of PRM by repressor bound at OR2 and by RNA polymerase bound at PR.

We have deleted a single base-pair in the -35 region of the bacteriophage lambda PRM promoter. The deletion (PRM delta 34) creates a better match of PRM to consensus, thereby substantially increasing the activity of the promoter in vitro and in vivo. Since the mutation also increases the overlap between OR2 and the -35 region of PRM, binding of repressor to OR2 no longer activates, but in fact represses PRM. Finally, the mutation decreases the distance between the PRM and PR transcription start sites from 82 to 81 base-pairs. As a consequence, the interaction of RNA polymerase with either promoter in vitro strongly inhibits open complex formation at the other. Kinetic analyses and DNase I protection assays lead to the surprising result that mutual inhibition is not due to steric occlusion. Both promoters can be occupied by RNA polymerase at the same time. Determination of KB and kf revealed that inhibition of PRM delta 34 by PR was manifest in a 100-fold decrease in the value of kf, but at the same time KB was increased tenfold. These data raise the possibility that RNA polymerase molecules bound at the two promoters contact and mutually stabilize each other and that this interaction subsequently inhibits a substep in the isomerization of closed to open complexes. In footprinting assays, each promoter is characterized by sites of enhanced cleavage when that promoter is occupied alone. These enhancements are substantially diminished when both promoters are occupied, suggesting that complexes of each promoter with RNA polymerase alter the structure of complexes formed at the other promoter. Assays of the effects of the delta 34 mutation in vivo indicate that interference between PRM and PR does not limit the rate of open complex formation at PRM in the cell. Apparently, transcription initiation clears the promoter rapidly enough that neither promoter is occupied a significant fraction of the time.

Nucleotide sequences of the trpI, trpB, and trpA genes of Pseudomonas syringae: positive control unique to fluorescent pseudomonads.

A 904-bp probe from Pseudomonas aeruginosa was used to identify the trpB, trpA and trpI genes of Pseudomonas syringae. Transcription initiation at the P. syringae trpBA promoter in vitro was activated by the P. aeruginosa TrpI protein in the presence of indoleglycerol phosphate. Thus, trpB and trpA are regulated positively in three species of fluorescent pseudomonads, P. aeruginosa, P. putida, and P. syringae, but in no other eubacteria so far investigated [Crawford, Annu. Rev. Microbiol. 43 (1989) 567-600]. In addition to conservation of protein-coding sequences, there is a high degree of nucleotide sequence identity in the intergenic control region that includes the divergent trpI and trpBA promoters, especially in the binding sites for TrpI protein. Differences in patterns of codon usage distinguish the trpI genes of P. syringae and P. putida from P. aeruginosa trpI and from the trpB and trpA genes of all three species.

Activation defects caused by mutations in Escherichia coli rpoA are promoter specific.

Escherichia coli RNA polymerases containing mutated alpha subunits were tested for their ability to respond to three different positive regulators (activators) in vitro. The two alpha (rpoA) mutants, alpha-256 and alpha-235, have deletions of the C-terminal 73 and 94 amino acids, respectively. In runoff transcription assays catalyzed by reconstituted holoenzyme, the effects of the mutations on each of three promoters tested were different: activation of the lambda pRM promoter by cI protein (repressor) was nearly normal, activation of the lambda pRE promoter by cII protein was reduced approximately fivefold, and direct activation of the trpPB promoter of Pseudomonas aeruginosa was completely inhibited. We also found that the reconstituted mutant enzyme was defective in recognition of trpPI in the absence of activator. The differential responses of the three promoters to their activators in the presence of the mutant enzymes indicate that the location of an activator-binding site does not by itself determine the region of RNA polymerase with which the activator interacts.

Mutations in TrpI binding site II that differentially affect activation of the trpBA promoter of Pseudomonas aeruginosa.

In vitro, Pseudomonas aeruginosa TrpI protein activates transcription initiation at the trpBA promoter (trpPB) and represses initiation at its own promoter (trpPI), which diverges from, and overlaps, trpPB. Indoleglycerol phosphate (InGP) reduces the TrpI concentration required for binding to its strong binding site (site I), as measured by repression of trpPI; it also facilitates activation of trpPB, presumably because it enables TrpI to bind to a weaker binding site (site II) and thereby interact with RNA polymerase. The role of site II and InGP in regulation of the two promoters was investigated by constructing site II mutants. A 2 bp substitution affected the ability of TrpI to activate trpPB, but did not significantly affect TrpI binding to site II. A more extensive (8 bp) substitution inhibited TrpI-mediated activation of trpPB and TrpI-mediated protection of site II in a DNase I footprinting assay. However, the mutation did not alter the pattern of TrpI binding observed in gel retardation experiments. In particular, a more slowly-migrating complex (Complex 2) whose appearance was correlated with TrpI binding to site II was formed equally well on a wild-type or substituted DNA fragment. Based on the mutant phenotypes, we propose that a particular sequence of protein--protein and protein--DNA interactions is required for activation of trpPB by TrpI and InGP.

Activation of the trpBA promoter of Pseudomonas aeruginosa by TrpI protein in vitro.

We have developed an in vitro transcription system in which purified TrpI protein and indoleglycerol phosphate (InGP) activate transcription initiation at the trpBA promoter (trpPB) and repress initiation at the trpI promoter (trpPI) of Pseudomonas aeruginosa. The phenotypes resulting from mutations in the -10 region of both promoters indicate that the -10 region consensus sequence in P. aeruginosa is probably the same as that in Escherichia coli. Furthermore, in the absence of TrpI and InGP, the activities of the two promoters are inversely correlated: down mutations in trpPI lead to increased activity of trpPB, and up mutations in trpPB cause a decrease in trpPI activity. These results are a consequence of the fact that the two promoters overlap, so that RNA polymerase cannot form open complexes with both promoters simultaneously. Thus, in theory, by preventing RNA polymerase from binding at trpPI, TrpI protein could indirectly activate trpPB. However, oligonucleotide-induced mutations that completely inactivate trpPI do not relieve the requirement for TrpI and InGP to activate trpPB. Therefore, activation of trpPB is mediated by a direct effect of TrpI on transcription initiation at trpPB. In addition, the oligonucleotide-induced mutations in trpPI alter site II, the weaker of two TrpI binding sites identified in DNase I and hydroxyl radical footprinting studies (M. Chang and I. P. Crawford, Nucleic Acids Res. 18:979-988, 1990). Since these mutations prevent full activation of trpPB, we conclude that specific base pairs in site II are required for activation.

RNA polymerases from Pseudomonas aeruginosa and Pseudomonas syringae respond to Escherichia coli activator proteins.

The activities of RNA polymerases (RNAPs) from Pseudomonas aeruginosa and Pseudomonas syringae were compared with that of Escherichia coli RNAP. All three enzymes are able to initiate transcription at the trpBA promoter of P. aeruginosa and at the coliphage lambda promoters, pRM and pRE, in response to heterospecific activators (TrpI protein, repressor, and cII protein, respectively). However, both Pseudomonas polymerases have less stringent requirements for promoter recognition in the absence of activators than does E. coli RNAP.

Characterization of a doubly mutant derivative of the lambda PRM promoter. Effects of mutations on activation of PRM.

The mutation, prmE37, located at -14 in the PRM promoter of bacteriophage lambda, reduces PRM function dramatically both in vitro and in vivo. In a search for second-site revertants of prmE37, we isolated a double mutant that exhibits a partially restored Prm+ phenotype. The second-site mutation (at -31) is identical to the mutation prmup-1. The activity of the doubly mutant (pseudo-revertant) promoter, prmE37prmup-1, was investigated in vivo using a PRM-lacZ fusion phage and found to be intermediate between that of prmE37 and wild-type PRM. However, the relative strength of the prmE37prmup-1 promoter was greater than expected following superinfection of a lambda lysogen. Since nalidixic acid was found to preferentially inhibit transcription from the doubly mutant promoter under these conditions, we suggest that DNA supercoiling favors activation of this promoter by repressor. In runoff transcription assays in the absence of repressor, the activity of wild-type PRM and the doubly mutant promoter were the same. However, while addition of repressor significantly stimulated wild-type PRM, it had little or no effect on the activity of the doubly mutant promoter. Values of KB, the equilibrium constant for formation of closed complexes, and kf, the rate constant for isomerization of closed to open complexes, were determined in abortive initiation assays, and the product of kfKB was used as a measure of promoter strength. The results of these assays are in agreement with those obtained in runoff transcription assays. In the absence of repressor, values of kfKB for the doubly mutant promoter and wild-type PRM are the same; however, tau obs, the time required for open complex formation, is significantly greater for the double mutant than for wild-type PRM at all RNA polymerase concentrations used for the abortive initiation analysis. In the presence of repressor, the doubly mutant promoter is stronger than the prmE37 promoter, but much weaker than wild-type PRM. This is due to the fact that kf for the doubly mutant promoter is increased 2.5-fold by repressor, but KB is reduced to the same extent. These two effects counteract each other, so that repressor has no net effect on the strength of the prmE37prmup-1 promoter in vitro. In contrast, repressor increases kf for wild-type PRM eightfold and increases overall promoter strength (KBkf) nearly fivefold. In the presence of repressor, the effects of the two mutations, prmE37 and prmup-1, on kf are independent. This observation is discussed in relation to revised models for open complex formation.

Interactions between Escherichia coli RNA polymerase and lambda repressor. Mutations in PRM affect repression of PR.

The rightward operator, OR, of bacteriophage lambda is part of a complex regulatory region that includes PRM, the promoter for repressor synthesis by a prophage, the rightward early promoter PR, and three repressor-binding sites, OR1, OR2 and OR3. By binding to OR2, repressor blocks transcription from PR and simultaneously stimulates the formation of open complexes between RNA polymerase and PRM. In this letter, we describe a test of the hypothesis that the interaction between RNA polymerase bound at PRM and repressor bound at OR2 increases the apparent affinity of repressor for OR. One implication of this hypothesis is that the amount of repressor required for repression of PR should be inversely correlated with PRM promoter strength. This is indeed the case. The amount of repressor required for 50% repression of PR is decreased by prmup-1, an "up" mutation of PRM, and is increased by prm- mutations. An unexpected finding is that in addition to their effect on the apparent affinity of repressor for OR, mutations in the -35 region of PRM alter the shape of repressor-titration curves. We propose that these mutations alter the interaction between RNA polymerase bound at PRM and repressor bound at OR2 in such a way that cooperativity in the binding of repressor to OR1 and OR2 is also disrupted.

Translational polarity of a mutation in the initiator AUG codon of the lambda cI gene.

A PRM-cI-lacZ fusion inserted into the b2 region of bacteriophage lambda was used to isolate mutations affecting expression of both the lambda cI gene and the lacZ gene. One such mutation, a change in the cI initiator codon from AUG to AUA, reduces immunity of a lambda prophage to superinfection, and causes a 60-70% reduction in beta-galactosidase synthesis, even when repressor is supplied in trans. The effect of the mutation on lacZ gene expression is eliminated in a rho- bacterial strain, and the mutation has no effect on transcription initiated at PRM in vitro. Therefore, the effects of the mutation are due to premature p-dependent termination of transcription in the absence of translation of the cI gene, as if the mutation were a nonsense polar mutation.