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PubMed · 6445470

Bacteriophage lambda repressor and cro protein: interactions with operator DNA.

A D Johnson·C O Pabo·R T Sauer
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Bacteriophage lambdaBase SequenceBinding SitesDNA, BacterialDNA-Binding ProteinsEscherichia coliKineticsOperonProtein BindingRepressor ProteinsTemperatureTranscription FactorsViral ProteinsViral Regulatory and Accessory ProteinsJournal Article

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A D Johnson, C O Pabo, R T Sauer. 1980. Bacteriophage lambda repressor and cro protein: interactions with operator DNA.. https://doi.org/10.1016/s0076-6879(80)65078-2

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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.

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PubMed1995

Kinetics of RNA polymerase initiation and pausing at the lambda late gene promoter in vivo.

We have measured the kinetics of transcription initiation and pausing by Escherichia coli RNA polymerase (RNAP) at the bacteriophage lambda late promoter, pR's, in growing cells. RNAP initiating transcription from pR' pauses after transcribing 16 or 17 nucleotides, and escape from this pause could in theory be the rate-limiting step in promoter function. We tested this hypothesis by analyzing pausing and non-pausing variants of both the pR' promoter segment and a more active mutant version of pR'; we measured reporter gene expression and used KMnO4 footprinting to measure directly occupancy of the promoter and pause sites in growing cells. We find that RNAP paused at +16/+17 does not limit expression of pR'. However, RNAP paused at +16/+17 does limit expression from the more active promoter by impeding formation of open complex. Therefore, the activity of the late gene regulatory protein Q to suppress the early pause, in addition to its antitermination activity, is unlikely to be important in phage gene expression.

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PubMed1995

HO. and DNase I probing of E sigma 70 RNA polymerase--lambda PR promoter open complexes: Mg2+ binding and its structural consequences at the transcription start site.

Chemical and enzymatic probing (footprinting) of the reactivity of the promoter DNA backbone is applied to characterize two binary open complexes RPo1 (-Mg2+) and RPo2 (+Mg2+), formed by Escherichia coli RNA polymerase (E sigma 70) at the lambda PR promoter. We report that HO. detects major differences in backbone reactivity between RPo1 and RPo2 in the open region from -4 to +1 relative to the transcription start site. Deoxyribose sugars at positions -4 to +1 of the t (template) strand react with HO. in RPo2 but are relatively protected in RPo1. Binding of Mg2+ to convert RPo1 to RPo2 therefore increases the reactivity of two negatively charged footprinting agents [MnO4-; Suh, W.-C., Ross, W., & Record, M. T., Jr., (1993) Science 259, 358-361; and Fe(EDTA)2-/HO.] at the start site and is required for binding of the negatively-charged initiating nucleotides to the polymerase and the t strand at the start site. We propose that these effects result from binding of two Mg2+ ions to the catalytic carboxyls in the nucleotide binding sites. Except for the key region on the t strand at the start site, the promoter DNA of both RPo1 and RPo2 is continuously protected from DNase I and hydroxyl radical (HO.) cleavage between the -12 and +25 promoter positions. Protection in the upstream region, extending from -13 to about -70, is periodic, with an 11 base pair periodicity indicative of binding of polymerase to a single face of the DNA helix.(ABSTRACT TRUNCATED AT 250 WORDS)

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