Interference of PR-bound RNA Polymerase with Open Complex Formation at PRM Is Relieved by a 10-Base Pair Deletion between the Two Promoters

Abstract

Bacteriophage promoters P R and P RM 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 P R promoter interfered with the utilization of the P RM 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 P RM . 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 P R and P RM . These findings suggest several ways in which for the wild type separation of the two promoters the utilization of P RM could be affected by an RNA polymerase at P R . In addition to direct steric interference, these include the obstruction of access to DNA sites necessary for optimal contact with the RNA polymerase.Promoters P R and P RM of bacteriophage direct the synthesis of nonoverlapping, divergent transcripts originating from start sites separated by 82 base pairs. We (1, 2) as well as Gussin and co-workers (3, 4) have shown that the presence of the P R promoter has a negative effect on the ability of RNA polymerase to form open complexes at P RM . The use of P R mutants allowed Gussin's group to demonstrate that conversely, the presence of an RNA polymerase at P RM negatively affects the utilization of a (weakened) P R promoter as well (5).Open complex formation at the wild type P R promoter occurs within seconds (6, 7), but formation of the complex at P RM is several orders of magnitude slower, on the time scale of tens of minutes (1,3,8). Thus nearly every RNA polymerase binding at P RM does so in the context of another polymerase situated at P R . Our own work (1, 2) as well as more extensive kinetic analysis by Gussin and his group (3, 4) has demonstrated that the interference with complex formation at P RM manifests itself not at the initial bimolecular (binding) step but rather in some subsequent step involving conformational transitions in RNA polymerase and/or DNA. The interference slows down but does not prevent open complex formation at P RM ; ultimately open complexes at both promoters do co-exist on the same DNA fragment (Refs. 1 and 9 and this work).Reduction of the distance between P R and P RM by one base pair further slows open complex formation at P RM (4). The effects of considerably shorter separation between the P R and P RM promoters can be assessed from studies on other lambdoid phages. For the 434 phage where the distance between the start sites of P R and P RM is about 65 base pairs, the Ϫ35 regions of the two promoters overlap and concurrent binding of RNA polymerases at the two promoters is not observed (10). For P22 the interpromoter distance is even shorter, making it quite unl...

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

“…Under favorable conditions, the distribution of proteins between several nucleic acid molecules can be monitored within a single solution 18,23 , as can the presence of complexes differing in protein stoichiometry and/or binding site distribution 7,24 . Proteins ranging in size from small oligopeptides to transcription complexes with M r ≥ 10 6 can give useful mobility shifts 25,26 and the assay works well with both highly-purified proteins and crude cell extracts 27 . These capabilities account in large part for the continuing popularity of the assay.…”

Promoter Interference in a Bacteriophage Lambda Control Region: Effects of a Range of Interpromoter Distances

“…Therefore, for the wild-type control region, in vitro RNA polymerase (RNAP)-p RM interactions occur almost exclusively in the context of another RNAP already bound to p R . It has been previously shown that this p R -bound RNAP interferes with open complex formation at p RM (16,17,21,34,37). The effect is not exerted at the initial binding of RNAP to the promoter but rather at a subsequent step (16, 34) that is likely a conformational change in the RNAP (9).…”

“…Consistent with this notion, deletion of 1 bp between the Ϫ35 regions was found to further reduce the rate of open complex formation at p RM (40). However, it has also been shown that when the distance between the Ϫ35 regions of the promoters is shortened by the deletion of 10 bp (one turn of the DNA helix), unexpectedly the inhibition of open complex formation at p RM is greatly diminished (21). In other phages where the interpromoter distance at p R and p RM is even shorter, such as 434 (66 pdb between start sites) and P22 (52 pdb), concurrent occupancy of the promoters is not observed (8,41).…”

“…Open complex formation at the p RM promoter was monitored with an electrophoretic mobility shift assay carried out as described by Mita et al (21). Approximately 1 to 2 nM 32 P-labeled promoter DNA was incubated at 37°C with RNAP (activity, 50% Ϯ 10% [mean Ϯ standard deviation]), at a concentration of active enzyme of 100 nM, in 20 l of HEPES buffer (30 mM HEPES [pH 7.6], 100 mM KCl, 10 mM MgCl 2 , 1 mM dithiothreitol) containing 50 g of bovine serum albumin per ml.…”

“…The constructs are designated as Dn, where n is the number of base pairs that have been deleted. The promoters were constructed from synthetic oligodeoxyribonucleotides and cloned into the pKK232-8 vector by using BamHI and HindIII restriction sites as described previously (21) and sequenced. The location of the strand-separated region at both promoters was checked by KMnO 4 footprinting and found not to be affected by the deletions (data not shown).…”