Differential effects of mutations on discrete steps in transcription initiation at the λ PRE promoter

Shih, Ming-Che; Gussin, Gary N.

doi:10.1016/0092-8674(83)90551-2

Cited by 37 publications

(17 citation statements)

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“…CRP, the cyclic AMP (cAMP) receptor protein, is an activator of many genes and operons and controls expression of lac, for example, by facilitating the binding of RNA polymerase (23). cI, A repressor, regulates its own expression by enhancing open complex formation at PRM (17,34). In this case as well, binding studies suggest a direct interaction (18).…”

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Suppressor mutations in rpoA suggest that OmpR controls transcription by direct interaction with the alpha subunit of RNA polymerase

1991

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We have isolated mutations in rpoA, the gene encoding the a subunit of RNA polymerase, that specifically affect transcriptional control by OmpR and EnvZ, the two-component regulatory system that controls porin gene expression in Escherichia coli. Characterization of these mutations and a previously isolated rpoA allele suggests that both positive and negative regulation of porin gene transcription involves a direct interaction between OmpR and RNA polymerase through the a subunit. Several of the rpoA mutations cluster in the carboxy-terminal portion of the a protein, further suggesting that it is this domain of a that is involved in interaction with OmpR and perhaps other transcriptional regulators as well.Several different mechanisms have been proposed to explain how regulatory proteins function to catalyze transcription initiation (1). A common feature of many of these models is a direct physical interaction between the activator protein and the transcription apparatus. Evidence supporting such an interaction has been presented for several prokaryotic regulatory proteins. CRP, the cyclic AMP (cAMP) receptor protein, is an activator of many genes and operons and controls expression of lac, for example, by facilitating the binding of RNA polymerase (23). cI, A repressor, regulates its own expression by enhancing open complex formation at PRM (17,34). In this case as well, binding studies suggest a direct interaction (18). NR1 (NtrC) is the effector of the two-component regulatory system that stimulates expression of the genes of the nitrogen regulon. It activates the glnAp2 promoter, for example, via a mechanism that involves DNA looping (40). A complete understanding of transcriptional regulation requires a more detailed description of these activator-RNA polymerase interactions. In the case of cI, genetic studies suggest a region of the repressor molecule that is involved in contacting RNA polymerase(6). However, in all cases, little is known about the oppos-ing contact region of the polymerase. We have sought to address this question in our studies of the two-component system that regulates porin gene expression in Escherichia coli.The two-component regulatory system, OmpR and EnvZ, controls the differential expression of the porin genes, ompF and ompC, in response to medium osmolarity. EnvZ is a receptor kinase that functions as a sensor of osmolarity and communicates this information to the effector, OmpR (an NR1 homolog), by a mechanism involving phosphorylation and dephosphorylation. OmpR-P controls transcription initiation by binding to sites upstream of the appropriate promoters (19). We have shown previously that OmpR works in a positive fashion at the ompC promoter and both positively and negatively at the ompF promoter (37, 38). In media of low osmolarity, levels of OmpR-P are low and * Corresponding author. ompF expression is activated. In media of high osmolarity, levels of OmpR-P increase, ompC expression is activated, and ompF expression is repressed (11). Mutations in envZ that result in increas...

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Suppressor mutations in rpoA suggest that OmpR controls transcription by direct interaction with the alpha subunit of RNA polymerase

1991

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“…Values of KB for the mutants were between one-fourth and one-third of the wild-type value. However, these effects are much smaller than the observed decreases in KB that result from mutations in the 35 consensus region itself (16).…”

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“…Thus, the T-T-G-C mutations differ substantially from mutations in the 35 consensus region, which decrease KB by factors of 20-140, and in some cases decrease k2 as well (16).…”

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“…We found that cy mutations at five sites within the two T-T-G-C sequences cause defects in the activation of PRE by cII protein but have little or no direct effect on the interaction between RNA polymerase and the promoter. These mutations differ significantly from other cy mutations, which affect the RNA polymerase-promoter interaction without affecting the binding of clI protein (16). A sixth T-T-G-C mutation, cy3001 (G --A at position -37), may affect the RNA polymerase-promoter interaction as WFell as cII binding.…”

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“…As we have shown (16,19), in the absence of clI protein, wild-type PRE is an extremely weak promoter, with KB = 1.0 x i7 M-1 and r = 42 min ( Table 2). Values of KB, T, and k2 for the mutant promoters (Table 2) were virtually the same as those obtained for wildtype PRE.…”

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Kinetic analysis of mutations affecting the cII activation site at the PRE promoter of bacteriophage lambda.

Shih¹,

Gussin²

1984

Proc. Natl. Acad. Sci. U.S.A.

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AbQrtive initiation and run-off transcription assays were used to study the effects of cy mutations on activation of the phage X PRE promoter by cI gene product. Six point mutations in the repeated T-T-G-C sequences that flank the -35 consensus region of PRE decreased the apparent affinity of the promoter for cIe protein by -factors of 4-16 relative to tjie wild-type affinity. Fig. 1) is that its -10 region agrees with the corresponding consensus promoter sequence (T-A-T-A-A-T) at only three of six sites, while its -35 region agrees with the, -35 consensus sequence (T-T-G-A-C-A) at only one site (8, 9). Nevertheless, mutations in PRE (CY mutations) have been found in both regions (11,12). In addition, cy mutations have been found in two T-T-G-C sequences that flank the -35 consensus region (Fig. 1). Since there are two T-T-G-C sequences at the identical position in P1 (13, 14), it was suggested that these sequences were required for clI protein binding. This prediction was confirmed by DNase protection experiments using wild-type and mutant templates (15).To investigate further the role of the T-T-G-C sequences in transcription initiation at PRE, we examined the effects of mutations in these sequences op activation of the promoter by cII protein in vitro. We found that cy mutations at five sites within the two T-T-G-C sequences cause defects in the activation of PRE by cII protein but have little or no direct effect on the interaction between RNA polymerase and the promoter. These mutations differ significantly from other cy mutations, which affect the RNA polymerase-promoter interaction without affecting the binding of clI protein (16). A sixth T-T-G-C mutation, cy3001 (G --A at position -37), may affect the RNA polymerase-promoter interaction as WFell as cII binding. MATERIALS AND METHODSPhage Strains. Phage X cy mutant strains (refs. 11 and 12; Fig. 1

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Transcription Activation Parameters at

Zhang

Reeder

Schleif

1996

Journal of Molecular Biology

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Differential effects of mutations on discrete steps in transcription initiation at the λ PRE promoter

Cited by 37 publications

References 19 publications

Suppressor mutations in rpoA suggest that OmpR controls transcription by direct interaction with the alpha subunit of RNA polymerase

Suppressor mutations in rpoA suggest that OmpR controls transcription by direct interaction with the alpha subunit of RNA polymerase

Kinetic analysis of mutations affecting the cII activation site at the PRE promoter of bacteriophage lambda.

Transcription Activation Parameters at

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