Bacteriophage P2 late promoters (original) (raw)

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.

Regulation of bacteriophage P2 late-gene expression: the ogr gene

Proceedings of the National Academy of Sciences of the United States of America, 1986

The ogr gene product of bacteriophage P2 is a positive regulatory factor required for P2 late-gene transcription. We have determined the nucleotide sequence of the ogr gene, which encodes a basic polypeptide of 72 amino acids. P2 growth is blocked by a host mutation, rpoA09, in the a subunit of DNA-dependent RNA polymerase. The ogr52 mutation, which allows P2 to grow in an rpoAl09 strain, was shown to be a single nucleotide change, in the codon for residue 42, that changes tyrosine to cysteine. The predicted amino acid sequence of the Ogr protein does not show similarity to DNAbinding proteins that are known to affect promoter recognition, to a factors, or to other characterized transcriptional regulatory proteins. We have inserted the ogr gene into a plasmid under control of the leftward promoter and operator of bacteriophage X. Thermal induction of ogr gene expression in this plasmid results in overproduction of a small protein that has been shown by complementation to possess Ogr function.

Identification of a Phage-coded DNA-binding Protein that Regulates Transcription from Late Promoters in Bacteriophage P4

Journal of Molecular Biology, 1996

The genetic element P4 can propagate as a temperate phage or as a Dipartimento di Genetica e di Biologia dei microrganismi multicopy plasmid in its host Escherichia coli. Late in the lytic cycle and in Università di Milano, Via the plasmid condition, transcription of the P4 essential genes depends on the activation of the late promoters P LL and P sid , which control the Celoria 26, 20133 Milano transcription of the left and right operons, respectively. Both P4 late Italy promoters are positively regulated by the product of the P4 d gene, which is transcribed from P sid . We have identified a new P4 gene, vis, that appears to play a relevant role in P4 late transcription control. vis is the first gene downstream of P LL and codes for a basic 88 amino acid protein with a potential helix-turn-helix motif. Expression of the cloned vis gene suppresses all the phenotypic traits exhibited by P4 vir1, a mutant that carries a promoter-up mutation in the late promoter P LL . By Northern hybridization analysis we showed that vis negatively regulates transcription from P LL and enhances transcription from P sid . Thus, vis auto-regulates its expression by repressing its own promoter and enhancing transcription of d, which is required for P LL activation.

The switch from early to late transcription in phage GA-1: characterization of the regulatory protein p4G

Journal of Molecular Biology, 1999

The transcription program of the Bacillus phage GA-1, a distant relative of phage È29, has been studied. Transcription of the GA-1 genome occurred in two stages, early and late. Early genes were expressed from two promoters equivalent to the È29 A2b and A2c promoters, whereas late transcription started at a site equivalent to the È29 late A3 promoter. The activity of the GA-1 early A2b and A2c promoters diminished 10 minutes after infection, a time at which expression of the late promoter increased signi®cantly. The switch from early to late transcription required protein synthesis, suggesting the need for viral protein(s). An open reading frame was found in the GA-1 genome coding for a protein showing a 53 % similarity to È29 regulatory protein p4, and was named p4 G . In È29, protein p4 represses the early A2b and A2c promoters and activates the late A3 promoter by recruiting RNA polymerase to it. A binding site for protein p4 G was localized upstream from the GA-1 late A3 promoter, overlapping with the early A2b promoter. In vitro, protein p4 G prevented the binding of RNA polymerase to the GA-1 early A2b promoter but, unlike in È29, had no effect on the expression of the late A3 promoter: RNA polymerase could ef®ciently bind and initiate transcription from the A3 promoter in the absence of protein p4 G . Therefore, activation of late transcription occurs differently in GA-1 and È29. We propose that protein p4 G is an anti-repressor which inhibits the binding to the late promoter of an unknown repressor factor present in the host strain.

Transcription of the T4 late genes

Virology Journal, 2010

This article reviews the current state of understanding of the regulated transcription of the bacteriophage T4 late genes, with a focus on the underlying biochemical mechanisms, which turn out to be unique to the T4-related family of phages or significantly different from other bacterial systems. The activator of T4 late transcription is the gene 45 protein (gp45), the sliding clamp of the T4 replisome. Gp45 becomes topologically linked to DNA through the action of its clamp-loader, but it is not site-specifically DNA-bound, as other transcriptional activators are. Gp45 facilitates RNA polymerase recruitment to late promoters by interacting with two phage-encoded polymerase subunits: gp33, the co-activator of T4 late transcription; and gp55, the T4 late promoter recognition protein. The emphasis of this account is on the sites and mechanisms of actions of these three proteins, and on their roles in the formation of transcription-ready open T4 late promoter complexes.

Activation of P2 late transcription by P2 ogr protein requires a discrete contact site on the C terminus of the α subunit of Escherichia coli RNA polymerase

Journal of Molecular Biology, 1997

Bacteriophage P2 late transcription requires the product of the P2 ogr gene. Ogr-dependent transcription from P2 late promoters is blocked by certain point mutations affecting the a subunits of the host RNA polymerase. An alanine scan spanning the putative activation target in the a C-terminal domain (aCTD) was carried out to identify individual residues essential for Ogr-dependent transcription from P2 late promoters. In addition, the effects of alanine substitutions in the regions of the aCTD previously reported to affect CAP-dependent activation of the lac promoter and UP-element DNA binding were examined. Residues E286, V287, L289 and L290 in helix 3, and residue L300 at the beginning of helix 4, de®ne a surface-exposed patch on the aCTD important for Ogr-dependent activation. These residues, adjacent to the recently identi®ed DNAbinding determinants, constitute a newly identi®ed activation surface for protein:protein contact. Alanine substitutions at some of the residues that affect UP-element DNA binding also impaired activation. This suggests that upstream DNA-a contacts, in addition to a-Ogr contacts, may be important in P2 late transcription. Other residues implicated in the interaction of a with CAP are not required for activation by Ogr, consistent with previous genetic evidence suggesting that these activators contact different sites on the aCTD.