Establishment of Lysogeny in Bacteriophage 186 (original) (raw)
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The Journal of biological chemistry, 2000
The CII protein of bacteriophage 186 is a transcriptional activator of the helix-turn helix family required for establishment of the lysogenic state. DNA binding by 186 CII is unusual in that the invertedly repeated half sites are separated by 20 base pairs, or two turns of the DNA helix, rather than the one turn usually associated with this class of proteins. Here, we investigate quantitatively the DNA binding properties of CII and its interaction with RNA polymerase at the establishment promoter, p(E). The stoichiometry of CII binding was determined by sedimentation equilibrium experiments using a fluorescein-labeled oligonucleotide and purified CII. These experiments indicate that the CII species bound to DNA is a dimer, with additional weak binding of a tetrameric species at high concentrations. Examination of the thermodynamic linkages between CII self-association and DNA binding shows that CII binds to the DNA as a preformed dimer (binding free energy, 9.9 kcal/mol at 4 degree...
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.
Specific contacts between the bacteriophage T3, T7, and SP6 RNA polymerases and their promoters
Journal of Biological Chemistry, 1991
The specificity and structural simplicity of the bacteriophage T3, T7, and SP6 RNA polymerases make these enzymes particularly well suited for studies of polymerase-promoter interactions. To understand the initial recognition process between the enzyme and its promoters, DNA fragments that carry phage promoters were chemically modified by three different methods: base methylation, phosphate ethylation, and base removal. The positions at which these modifications prevented or enhanced binding by the RNA polymerases were then determined. The results indicate that specific contacts within the major groove of the promoter between positions-5 and-12 are important for phage polymerase binding. Removal of individual bases from either strand of the initiation region (-5 to +3) resulted in enhanced binding of the polymerase, suggesting that disruption of the helix in this region may play a role in stabilization of the polymerasepromoter complexes.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1993
Using a rifampicin-resistant RNA polymerase with altered specificity to different promoters, the D promoter of T7 phage DNA with increased affinity to the mutant enzyme was chosen. This promoter and the T7 A1 promoter with unchanged affinity as well as some nonpromoter DNA fragments were used to compare temperature-induced conformational transitions of RNA polymerase in the course of complex formation. Conformational alterations of RNA polymerase were monitored by the fluorescent label method. It was shown that RNA polymerase undergoes a set of conformational transitions during complex formation with each promoter, some of which were similar by the character of change to spectral parameters of the label (reflecting RP i and, probably, RP o formation). The local structure of complexes formed above 33°C differs for A1 and D. The conformational analysis reveals at least one temperature-dependent stage upon nonspecific interaction of the enzyme with nonpromoter DNA at 13-16°C. Models of functional organization of the enzyme recognizing center and some features of the structure of the promoters which may be essential for their recognition are discussed.
Frontiers in Molecular Biosciences, 2024
Transcription initiation is a multi-step process, in which the RNA polymerase holoenzyme binds to the specific promoter sequences to form a closed complex, which, through intermediate stages, isomerizes into an open complex capable of initiating the productive phase of transcription. The aim of this work was to determine the contribution of the −10 and −35 regions of the promoter, as well as the role of non-specific interactions, in the binding of RNA polymerase and the formation of an active initiation complex capable of transcription. Therefore, fragments of promoter DNA, derived from the strong promoter A1 of the phage T7, containing completely and partially altered elements −35 and −10, and devoid of an upstream region, were constructed using genetic engineering methods. Functional analyses of modified promoter fragments were carried out, checking their ability to form binary complexes with Escherichia coli RNA polymerase (RNAP) and the efficiency of converting binary complexes into triple complexes characteristic of the productive phase of transcription. The obtained results suggest that, in relation to the A1 promoter of the T7 phage, the most important role of the −35 region is carrying the open complex through the next phases of transcription initiation. The weakening of specific impacts within the region −35 is a reason for the defect associated with the transformation of the open complex, formed by a DNA fragment containing the completely altered −35 region, into elongation and the impairment of RNA synthesis. This leads to breaking contacts with the RNA polymerase holoenzyme, and destabilization and disintegration of the complex in the initial phase of productive transcription. This confirms the hypothesis of the so-called stressed intermediate state associated with the stage of transition from the open complex to the elongation complex. The experiments carried out in this work confirm also
FEMS Microbiology Letters, 1996
It was demonstrated previously that a mutation, rpoA341, in the gene encoding the a subunit of Escherichiu coli RNA polymerase prevents lysogenization by bacteriophage h. The rpoA341 allele is known to be responsible for impaired transcription of some positively regulated E. coli chromosomal operons. Here we demonstrate that the inhibition of lysogenization of the rpoA341 mutant is a result of drastically decreased transcription from positively regulated phage promoters. We were unable to detect any transcripts originating from the CII-activated pz, pi and paQ promoters (important for lysogenic development) in, the phage-infected rpoA341 mutant, in contrast to an otherwise isogenic rpoA+ strain. The results are discussed in the light of other reports showing that activation of the pa promoter by CII protein in vitro is decreased only about fivefold when the native a subunit is replaced by truncated a polypeptides.
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.
Molecular Microbiology, 1996
Phage φ29 regulatory protein p4 activates transcription from the late A3 promoter by stabilizing σA-RNA polymerase at the promoter as a closed complex. Activation requires interaction between both proteins. Protein p4 bends the DNA upon binding. We have performed a detailed mutagenesis study of the carboxyl end of the protein, which is involved in both transcription activation and DNA bending. The results indicate that Arg-120 is the most critical residue for activation, probably mediating the interaction with RNA polymerase. Several basic residues have been identified, including Arg-120, that contribute to maintenance of the DNA bending, probably via electrostatic interactions with the DNA backbone. The degree or stability of the induced bend apparently relies on the additive contribution of all basic residues of the carboxyl end of the protein. Therefore, the activation and DNA bending surfaces overlap, and Arg-120 should interact with both DNA and RNA polymerase. As we show that protein p4 is a dimer in solution, and is bound to DNA as a tetramer, the results suggest a model in which two of the p4 subunits interact with the DNA, bending it, while the other two subunits remain accessible to interact with RNA polymerase.