Direct and Indirect Effects of Mutations in λ PRM on Open Complex Formation at the Divergent PR Promoter

Fong, Raymond Shiaw-Ching; Woody, Scott; Gussin, Gary N.

doi:10.1006/jmbi.1994.1426

Cited by 16 publications

(12 citation statements)

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“…The existence of upstream interactions at p R is consistent with published footprinting results, which show protection in this region (5). In further support of such interactions at p R , an A-T substitution for a G-C base pair at position Ϫ51, resulting in an uninterrupted ATR of 12 bp, has been found to lead to a threefold-increased rate of open complex formation at p R (7). Both the latter observation and our own that p R 's activity, but not that of p RM , is sensitive to alanine substitutions in the ␣ subunit are suggestive of some sequence-specific recognition of the (AϩT) base pairs in p R 's ATR.…”

Section: Atrsupporting

confidence: 87%

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

et al. 1996

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The rightward regulatory region of bacteriophage lambda contains two promoters, p RM and p R , 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 p RM 's ATR. We determined the effects on RNA polymerase-p RM promoter interaction both of (G؉C) substitutions in the ATR and of amino acid substitutions in the ␣ subunit, known to affect the upstream interaction. We find small (two-to threefold) effects of selected mutations in the ␣ subunit on open complex formation at p RM . However, the (presumably upstream) interactions underlying these effects are sequence nonspecific, as they are not affected by ( The rightward regulatory region of bacteriophage lambda contains two promoters, p R and p RM , which are divergently transcribed from start sites separated by only 82 bp (25). Open complex formation at the p R promoter is much faster than at p RM ; therefore, the vast majority of RNA polymerases (RNAPs) forming an open complex at the latter promoter do so on a fragment that already contains a polymerase at p R . It was found by us (14, 15) as well as by Gussin's group (6, 28) that mutations which inactivate the p R promoter facilitate open complex formation at p RM . These results suggest that p R -bound RNAP interferes with open complex formation at p RM . Surprisingly, a 10-bp deletion between the two promoters appears to abolish the interference (22), as it results in increased activity of p RM whether p R is inactivated by mutation or not.The identity of promoters for Escherichia coli RNAP is mostly determined by two DNA elements, the Ϫ10 and Ϫ35 regions (21). The ability to recognize these two regions is imparted upon the RNAP holoenzyme by its subunit (12). The promoters for E. coli rRNA (27), as well as some other strong promoters in E. coli (17)(18)(19) and Bacillus subtilis (2, 13), have been found to additionally have (AϩT)-rich regions (ATRs) upstream of bp Ϫ40. These so-called UP elements (27) are typically 20 bp in length. Transcription in vivo and in vitro (27) as well as the rate of open complex formation at the rrnB P1 promoter (26) was decreased by more than 10-fold because of deletion of this UP element. The C-terminal domain (amino acids 249 to 329 [3,23]) of the ␣ subunit of RNAP has been implicated in UP element recognition: deletion of this domain greatly impaired the ability of RNAP to respond to the UP element both in vivo and in vitro (27). In subsequent studies, amino acid substitutions in the ␣ subunit were employed to pinpoint the amino acids involved in the interaction with the UP element to those at positions 262 to 269 and 296 to 299 (8). Recent determinations of the structure of the Cterminal domain of the ␣ subunit show that the location of these amino acids is compatible wit...

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Section: Atrsupporting

confidence: 87%

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

et al. 1996

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“…P R is slightly stronger when the region from Ϫ66 to Ϫ42 is present than in its absence (44), although this region does not have this effect in a different context. An AT-rich segment within this region matches fairly well with the upstream part of a consensus UP element (44), and a P RM mutation, prm116, in the Ϫ35 region that improves this match is known to increase the strength of P R (9). The region changed here in the Ϫ35 region of P RM is included in that AT-rich segment, and the match is weakened in the nine variants analyzed.…”

Section: Discussionmentioning

confidence: 78%

Sequence Tolerance of the Phage λ P _RM Promoter: Implications for Evolution of Gene Regulatory Circuitry

Michalowski¹,

Short²,

Little

2004

J Bacteriol

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Much of the gene regulatory circuitry of phage centers on a complex region called the O R region. This ϳ100-bp region is densely packed with regulatory sites, including two promoters and three repressor-binding sites. The dense packing of this region is likely to impose severe constraints on its ability to change during evolution, raising the question of how the specific arrangement of sites and their exact sequences could evolve to their present form. Here we ask whether the sequence of a cis-acting site can be widely varied while retaining its function; if it can, evolution could proceed by a larger number of paths. To help address this question, we developed a cloning vector that allowed us to clone fragments spanning the O R region. By using this vector, we carried out intensive mutagenesis of the P RM promoter, which drives expression of CI repressor and is activated by CI itself. We made a pool of fragments in which 8 of the 12 positions in the ؊35 and ؊10 regions were randomized and cloned this pool into the vector, making a pool of P RM variant phage. About 10% of the P RM variants were able to lysogenize, suggesting that the regulatory circuitry is compatible with a wide range of P RM sequences. Analysis of several of these phages indicated a range of behaviors in prophage induction. Several isolates had induction properties similar to those of the wild type, and their promoters resembled the wild type in their responses to CI. We term this property of different sequences allowing roughly equivalent function "sequence tolerance " and discuss its role in the evolution of gene regulatory circuitry.Complex gene regulatory circuits can have a large number of interlocking components. This degree of interconnectivity raises two issues. First, how did these circuits evolve? Second, how can we understand the behavior of these existing circuits and predict their behavior in the face of small changes in parameters such as promoter strength? For these and other reasons, we have been analyzing the behavior of the regulatory circuitry of phage in the intact system. This system is probably the best-understood complex circuit (38, 39). Most, if not all, of the regulatory interactions have been identified, and most of these are well characterized at the mechanistic level. Previous analysis of this circuit generally has been carried out in uncoupled systems (such as the use of reporter genes and fusions with the lac promoter), an approach necessary to disentangle the causality of this system. With a circuit diagram in hand, it is now possible to return to the intact circuit and ask how particular changes affect the overall operation of the system.Many of the critical interactions in the circuit center on a complex regulatory region termed the O R region (Fig. 1C). This ϳ100-bp region is densely packed with cis-acting sites, including two promoters and three sites to which both the CI and Cro repressors can bind (38). In addition, the promoters and repressor-binding sites overlap extensively. CI and Cro regulate the expr...

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“…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).…”

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confidence: 99%

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

Mita

Tang

deHaseth

1995

Journal of Biological Chemistry

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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...

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Direct and Indirect Effects of Mutations in λ PRM on Open Complex Formation at the Divergent PR Promoter

Cited by 16 publications

References 0 publications

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

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

Sequence Tolerance of the Phage λ P _RM Promoter: Implications for Evolution of Gene Regulatory Circuitry

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

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Direct and Indirect Effects of Mutations in λ PRM on Open Complex Formation at the Divergent PR Promoter

Cited by 16 publications

References 0 publications

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

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

Sequence Tolerance of the Phage λ P RM Promoter: Implications for Evolution of Gene Regulatory Circuitry

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

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Sequence Tolerance of the Phage λ P _RM Promoter: Implications for Evolution of Gene Regulatory Circuitry