A cryptic promoter in the O(R) region of bacteriophage lambda (original) (raw)
Related papers
1993
A cryptic promoter, designated P.,D initiates transcription within the OR region of bacteriophage A. Transcription from P. proceeds in the direction of the cI repressor gene from sites 46 and 48 bp preceding the PRM transcription start site. P, 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, is blocked by A repressor. Kinetic analysis of relative abundance of the products of in vitro transcription indicated that P, was approximately 1/3 as strong as PRM. However, a P0-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,. are relatively unstable (half-life = 20 to 25 min). Dissociation of RNA polymerase from P., allows additional open complexes to form at PR or PRM, and thus the apparent strength of P,. d...
Journal of Molecular Biology, 1991
We demonstrate that RNA polymerase bound at the PR promoter of bacteriophage lambda can repress transcription initiation from the divergently transcribed PRM promoter in vitro. Using abortive initiation and run-off transcription experiments we show that inactivating mutations introduced into either the -10 or -35 regions of Pa result in a significant increase in the rate of formation of transcriptionally competent complexes at the PRM promoter. This is due primarily to an increase in the rate constant for the isomerization of closed to open complexes. Gel shift and DNase I footprinting experiments were employed to further define the mechanism by which Pa sequences mediate PRM repression. From these assays we were able to conclude that the formation of an open complex at the Pa promoter did not exclude RNA polymerase from binding at PRM. Rather, initiation at PRM was impaired because closed complexes must isomerize in the presence of an open complex already situated at the Pa promoter. Extensive evidence has been obtained previously indicating that lambda repressor activates transcription directly by contacting RNA polymerase situated at the PRM promoter. Results presented here raise the possibility that an additional mechanism could be operative, whereby lambda repressor indirectly activates PRM transcription by excluding RNA polymerase from the Pz promoter.
Selection for mutations in the P R promoter of bacteriophage lambda
Nucleic Acids Research, 1990
Insertion of DNA containing P R , the early rlghtward promoter of bacteriophage lambda, Is lethal to M13-derived vectors when the promoter directs transcription (using the ' +' strand as template) toward the M13 origin of replication (oil). Lethality can be relieved by mutation of P R , repression of the promoter by the X c\ repressor, or by insertion of a strong transcription terminator between P R and ori. We have used selection for plaque formation in the absence of repressor to isolate 14 different mutations at 8 sites In P R. This method of isolating promoter mutants In vivo is applicable generally to strong promoters whose activity is regulated either positively or negatively.
Journal of Bacteriology, 2000
The p R and p RM promoters of bacteriophage lambda direct transcription in divergent directions from start sites separated by 83 phosphodiester bonds. We had previously shown that the presence of an RNA polymerase at p R interfered with open complex formation at p RM and that this effect was alleviated by the deletion of 10 bp between the two promoters. Here we present a detailed characterization of the dependence of the interference on the interpromoter distance. It was found that the reduced interference between the two promoters is unique to the 10-bp deletion. The relief of interference was demonstrated to be due to the facilitation of a step subsequent to RNA polymerase binding to the p RM promoter. A model to explain these observations is proposed. A search of known Escherichia coli promoters identified three pairs of divergent promoters with similar separations to those investigated here.
Nucleic Acids Research, 2004
The bacteriophage l CII protein stimulates the activity of three phage promoters, p E , p I and p aQ , upon binding to a site overlapping the ±35 element at each promoter. Here we used preparations of RNA polymerase carrying a DNA cleavage reagent attached to speci®c residues in the C-terminal domain of the RNA polymerase a subunit (aCTD) to demonstrate that one aCTD binds near position ±41 at p E , whilst the other aCTD binds further upstream. The aCTD bound near position ±41 is oriented such that its 261 determinant is in close proximity to s 70 . The location of aCTD in CII-dependent complexes at the p E promoter is very similar to that found at many activator-independent promoters, and represents an alternative con®guration for aCTD at promoters where activators bind sites overlapping the ±35 region. We also used an in vivo alanine scan analysis to show that the DNA-binding determinant of aCTD is involved in stimulation of the p E promoter by CII, and this was con®rmed by in vitro transcription assays. We also show that whereas the K271E substitution in aCTD results in a drastic decrease in CII-dependent activation of p E , the p I and p aQ promoters are less sensitive to this substitution, suggesting that the role of aCTD at the three lysogenic promoters may be different.
Nucleic Acids Research, 2009
The stringent response effector, guanosine tetraphosphate (ppGpp), adjust gene expression and physiology in bacteria, by affecting the activity of various promoters. RNA polymerase-interacting protein, DksA, was proposed to be the co-factor of ppGpp effects; however, there are reports suggesting independent roles of these regulators. Bacteriophage j major lytic promoter, pR, is down-regulated by the stringent response and ppGpp. Here, we present evidence that DksA significantly stimulates pR-initiated transcription in vitro in the reconstituted system. DksA is also indispensable for pR activity in vivo. DksAmediated activation of pR-initiated transcription is predominant over ppGpp effects in the presence of both regulators in vitro. The possible role of the opposite regulation by ppGpp and DksA in j phage development is discussed. The major mechanism of DksA-mediated activation of transcription from pR involves facilitating of RNA polymerase binding to the promoter region, which results in more productive transcription initiation. Thus, our results provide evidence for the first promoter inhibited by ppGpp that can be stimulated by the DksA protein both in vivo and in vitro. Therefore, DksA role could be not only independent but antagonistic to ppGpp in transcription regulation.
Identification of Upstream Sequences Essential for Activation of a Bacteriophage P2 Late Promoter
Journal of Bacteriology, 2003
We have carried out a mutational scan of the upstream region of the bacteriophage P2 FETUD late operon promoter, P F , which spans an element of hyphenated dyad symmetry that is conserved among all six of the P2 and P4 late promoters. All mutants were assayed for activation by P4 Delta in vivo, by using a lacZ reporter plasmid, and a subset of mutants was assayed in vitro for Delta binding. The results confirm the critical role of the three complementary nucleotides in each half site of the upstream element for transcription factor binding and for activation of transcription. A trinucleotide DNA recognition site is consistent with a model in which these transcription factors bind via a zinc finger motif. The mutational scan also led to identification of the ؊35 region of the promoter. Introduction of a 70 ؊35 consensus sequence resulted in increased constitutive expression, which could be further stimulated by Delta. These results indicate that activator binding to the upstream region of P2 late promoters compensates in part for poor 70 contacts and helps to recruit RNA polymerase holoenzyme.
Deletion analysis of a bacteriophage P2 late promoter
Gene, 1990
We have fused the promoter (PF) for the P2 late FETUD oceron to the gene (cat) encoding chloramphenicol acetyltransferase (CAT) in a plasmid vector. Synthesis of CAT in Escherichia coli strains carrying this plasmid requires the product of the P2 ogr gene or the satellite phage P4 transactivation gene, 6. Our results demonstrate that these phage-encoded transcriptional regulatory proteins are necessary and sufficient for activation of P2 late transcription in this reporter plasmid. Positive regulation of cloned PF is severely impaired in a host strain carrying the rpod 109 mutation. Expression from the cloned promoter thus approximates those features of P2 late transcription that have been shown to occur during normal P2 infection. To define sequences required for promoter function, sequential upstream deletions of Pv were generated using BAL 31 nuclease, and the mutant promoters were assayed for cat expression. A sequence between nucleotides-69 and-64 from the transcription start point was found to be essential for promoter activity. This coincides with a region of homology conserved among all four P2 late gene promoters and the two P4 late promoters, and includes an element of dyad symmetry.
On the control of transcription of bacteriophage Mu
MGG Molecular & General Genetics, 1974
The transcription pattern of bacteriophage Mu has been studied with the use of Mu-lcts62, a thermo-inducible derivative of wild-type Mu. The rate of transcription at various times after induction was measured by pulse-labeling the RNA during synthesis and determining the fraction of Mu-specific RNA by hybridization with the separated strands of Mu-DNA. Transcription was found to take place predominantly from the heavy strand of Mu-DNA, as was found previously by Bade (1972). A study of the kinetics of this process revealed four phases. Initially after the induction the rate of transcription increases and reaches a maximum after four minutes. In the second phase during five minutes the rate falls down. During the third phase, up to 25 minutes after induction, the rate of transcription rises slowly, followed by a very rapid increase in the final phase, at the end of the lytic cycle. Phage Mu can be integrated in the host chromosome in two opposite orientations. The strand specificity, rate and time-course of transcription appeared not to be influenced by the orientation. The presence of chloramphenicol during the induction of the phage does not have an effect on the initial phase of transcription, but it prevents the decrease in the second phase. This suggests that in the early phase a Mu-speeific protein is synthesized which acts as a negative regulator of trancription. In non-permissive strains, lysogenic for a phage with an amber mutation in gene A or B, the transcription during the first and the second phase is the same as with wild-type phage; in the third phase, however, there is much less transcription than with wild type phage, whereas in the final phase the increase of the transcription rate is completely absent. Control experiments showed that DNA synthesis does not take place when a non-permissive strain is infected with a phage with an amber mutation in gene A or B. Therefore we conclude that the products of the genes A and B are required, directly or indirectly, for the autonomous replication of phage DNA. Since these amber mutants arc also impaired in the integration process, we conclude that the genes A and B code for regulator proteins with a crucial role in the development of bacteriophage Mu.