Protein p4 represses phage phi 29 A2c promoter by interacting with the alpha subunit of Bacillus subtilis RNA polymerase (original) (raw)
Related papers
Journal of Molecular Biology, 1998
Regulatory protein p4 from Bacillus subtilis phage È29 represses the early A2c promoter by binding upstream from RNA polymerase and interacting with the C-terminal domain of the RNA polymerase a subunit. This interaction stabilizes the RNA polymerase at the promoter in such a way that promoter clearance is prevented. Here, the binding of protein p4 to the A2c promoter has been studied. In the absence of RNA polymerase, protein p4 was found to bind with low af®nity to a site centered at position À39 relative to the transcription start site. When RNA polymerase was present, protein p4 was displaced from this site and bound instead to a different target centered at position À71. Stable binding to this site requires the interaction of protein p4 with the C-terminal domain of the RNA polymerase a-subunit. Both sites contain sequences resembling the well-characterized p4 binding site present at the late A3 promoter, to which p4 binds with high af®nity. A mutational analysis revealed that the site at À71 is critical for a stable interaction between protein p4 and RNA polymerase, and for ef®cient repression, whereas mutation of the site at À39 had only a small effect on repression ef®ciency. Therefore, RNA polymerase plays an active role in the repression mechanism by stabilizing the repressor at the promoter, generating a nucleoprotein complex that is too stable to allow promoter clearance.
Journal of Molecular Biology, 2001
Regulatory protein p4 from Bacillus subtilis phage f29 activates the viral late A3 promoter mainly by stabilizing the binding of RNA polymerase (RNAP) to it as a closed complex. This requires an interaction between protein p4 residue Arg120 and the C-terminal domain (CTD) of the RNAP a subunit. Several acidic residues of the a-CTD, considered as plausible targets for p4 residue Arg120, were individually changed into alanine. In addition, a truncated a subunit lacking the last four residues, two of which are acidic, was obtained. The modi®ed a subunits were puri®ed and reconstituted into RNAP holoenzyme in vitro. Protein p4 was found to be unable to activate the late A3 promoter when residue Glu297 of the a subunit was changed to Ala, a modi®cation that did not impair transcription from several other promoters. Interestingly, protein p4 could stabilize the modi®ed RNAP at the A3 promoter as a closed complex, although the open complexes formed were unstable and did not proceed to elongation complexes. Our results indicate that the change of the a residue Glu297 into Ala destabilizes the open complexes formed at this promoter, but not at other promoters. Considered in the context of earlier ®ndings indicating that the RNAP a-CTD may participate in the transition from closed to intermediate complexes at some other promoters, the new results expand and clarify our view of its role in transcription initiation.
The EMBO Journal, 1996
Phage 029 regulatory protein p4 activates transcription from the late A3 promoter and represses the main early promoters, named A2b and A2c. Activation involves stabilization of RNA polymerase (RNAP) at the A3 promoter as a closed complex and is mediated by interaction between RNAP and a small domain of protein p4 in which residue Argl20 plays an essential role. We show that protein p4 represses the A2c promoter by binding to DNA immediately upstream from RNAP in a way that does not hinder RNAP binding; rather, the two proteins bind cooperatively to DNA. In the presence of protein p4, RNAP can form an initiated complex at the A2c promoter that generates short abortive transcripts, but cannot leave the promoter. Mutation of protein p4 residue Argl20, which relieves the contact between the two proteins, leads to a loss of repression. Therefore, the contact between protein p4 and RNAP through the protein p4 domain containing Argl20 can activate or repress transcription, depending on the promoter. The relative position of protein p4 and RNAP, which is different at each promoter, together with the distinct characteristics of the two promoters, may determine whether protein p4 activates or represses transcription.
Journal of Molecular Biology, 1998
Regulatory protein p4 of Bacillus subtilis phage È29 activates transcription from the viral late A3 promoter by interacting with the C-terminal domain (CTD) of the B. subtilis RNA polymerase a subunit, thereby stabilizing the holoenzyme at the promoter. Protein p4 does not interact with the Escherichia coli RNA polymerase and cannot activate transcription with this enzyme. We have constructed a chimerical a subunit containing the N-terminal domain of the E. coli a subunit and the CTD of the B. subtilis a subunit. Reconstitution of RNA polymerases containing this chimerical a subunit, the E. coli b and b H subunits, and the vegetative s factor from either E. coli (s 70) or B. subtilis (s A), generated hybrid enzymes that were responsive to protein p4 and ef®ciently supported activation at the A3 promoter. Protein p4 activated transcription with the chimerical enzymes through the same activation surface used with B. subtilis RNA polymerase. Therefore, the B. subtilis a-CTD allowed activation by p4 even when the rest of the RNA polymerase subunits belonged to E. coli, a distantly related bacterium. These results strongly suggest that protein p4 works essentially by serving as an anchor that stabilizes RNA polymerase at the 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.
Journal of Molecular Biology, 1990
Transcription initiation from the Bacillus subtilis phage 429 late A3 promoter requires the viral protein p4, a transcriptional activator. Protein p4 binds to a region of the A3 promoter, located between nucleotides -50 and -100 relative to the transcription start site, that presents a sequence-directed curvature. This curvature is enhanced when protein p4 binds to the promoter. A number of deletion mutants at the carboxyl end of protein p4 have been constructed and their behavior as transcriptional activators of the late A3 promoter has been investigated. The binding of these deletion mutants to the late A3 promoter has been analyzed by gel retardation, DNase I footprinting, methylation interference and circular permutation assays. The results suggest that the last 12 amino acid residues of protein ~4, six of which are positively charged, although not involved in the specific recognition of the promoter are responsible for part of the bend induced by protein p4 in its binding site. Evidence is presented which suggests that full induction of this curvature is needed for the transcription activation process. A model is proposed for protein p4 interaction with the A3 promoter, in which the bend is induced in two steps: first, two monomers of protein p4 bind to the inverted recognition sequences, subsequent interaction between them generating a bend between these sequences; second, the highly basic carboxyl terminus of protein p4 establishes non-specific electrostatic interactions with the DNA backbone inducing a bend at both ends of the protein p4 binding region.
Initiation of the transcription of Φ29 DNA by Bacillus subtilis RNA polymerase
Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1974
The initiation of the transcription of ~b29 DNA by Bacillus subtilis RNA polymerase, in the presence or absence of o factor, takes place with GTP and to a lesser extent with ATP. In spite of the similarity of the initiation with purines, the RNA products are essentially asymmetric when the a factor is present and partially symmetric in its absence. Several dinucleoside monophosphates stimulate the transcription of ~b29 DNA by the B. subtilis RNA polymerase holoenzyme at a low concentration of nucleoside triphosphates. The dinucleotides U--A, C--A and A--U compete with the incorporation of [7-32 p] ATP to the 5' end of the RNA chains and U--G,
Gene, 1986
~~ciZZ#s s~~iiZis phage (p29 main promoters are effkiently recognized in vivo by the ~tre~~~~~~~s iividans RNA potymerase (Recombinant DNA; bacteriophage promoters; S 1 mapping; heterologous expression) SUMMARY A DNA fragment from the Bacillus subtilis phage 29,containingthemainearlyandlateviralpromoters,hasbeeninsertedupstreamoftheaminoglycosidephosphotransferasegene(nea)derivedfromthetransposonTn5andpresentinaStreptomyceslividanspromoter−probeplasmid.The29, containing the main early and late viral promoters, has been inserted upstream of the aminoglycoside phosphotransferase gene (nea) derived from the transposon Tn5 and present in a Streptomyces lividans promoter-probe plasmid. The 29,containingthemainearlyandlateviralpromoters,hasbeeninsertedupstreamoftheaminoglycosidephosphotransferasegene(nea)derivedfromthetransposonTn5andpresentinaStreptomyceslividanspromoter−probeplasmid.The29 promoters are specifically recognized by the S'. Zividans RNA polymerase which initiates transcription in vivo at the same sites utilized in B. su~ti~is. Moreover, the viral promoters efficiently direct the synthesis of high levels of the APHII enzyme in S. lividans.