Deletion analysis of a bacteriophage P2 late promoter

Grambow, Nancy J.; Birkeland, Nils Kåre; Anders, Douglas L.; Christie, Gail E.

doi:10.1016/0378-1119(90)90407-i

Cited by 31 publications

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References 26 publications

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“…64,65 Control of late gene expression P2 late promoters share a conserved DNA sequence that is centered 55 base pairs upstream of the transcription start 66,67 and required for late gene expression. 68,69 These sequences are recognized and bound by the 72 amino acid P2 Ogr protein, a zinc-binding transcription factor 70 which interacts with the C-terminal domain of the a subunit of E. coli RNA polymerase to stimulate late transcription. 71,72 The ogr gene is expressed at middle times after infection from its own promoter and also cotranscribed with the FETUD gene cluster late in infection.…”

Section: The Lysis Genesmentioning

confidence: 99%

Bacteriophage P2

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2016

Bacteriophage

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Section: The Lysis Genesmentioning

confidence: 99%

Bacteriophage P2

Christie

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2016

Bacteriophage

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“…The satellite and helper classes of activators also show differences in promoter specificity, as assayed in vivo with reporter plasmids. The helper phage activators are generally less efficient on their own promoters and show better expression from the P4 late promoter P sid (1,14), while the satellite phage activators work more efficiently at the helper phage late promoters (1,10,14). Since NucC activates transcription from chromosomal promoters in S. marcescens, we expected that it would fall into the satellite class of activators.…”

Section: Resultsmentioning

confidence: 99%

“…Expression of the activator was induced by shifting the culture to 42°C. Cells were harvested 30 min after induction, and CAT activity was measured spectrophotometrically as described previously (10).…”

Section: Purification Of Nucc Proteinmentioning

confidence: 99%

“…Four invariant Cys residues, arranged in a Cys-X 2 -Cys-X 22 -Cys-X 4 -Cys motif, are essential for function and involved in the coordination of zinc (8,15,19,21,26). Binding sites for these proteins have been localized upstream of P2 and P4 late promoters and the S. marcescens nuclease promoter (1,10,13,14,33,34); these sites include a conserved element of hyphenated dyad symmetry, TGT-N 12 -ACA. Genetic evidence indicates that members of this family of proteins activate transcription via a specific interaction with the C-terminal domain of the ␣ subunit of RNA polymerase (2,31,35).…”

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Purification and In Vitro Characterization of the Serratia marcescens NucC Protein, a Zinc-Binding Transcription Factor Homologous to P2 Ogr

et al. 2003

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NucC is structurally and functionally homologous to a family of prokaryotic zinc finger transcription factors required for late gene expression in P2-and P4-related bacteriophages. Characterization of these proteins in vitro has been hampered by their relative insolubility and tendency to aggregate. We report here the successful purification of soluble, active, wild-type NucC protein. Purified NucC exhibits site-specific binding to a conserved DNA sequence that is located upstream of NucC-dependent Serratia marcescens promoters and the late promoters of P2-related phages. This sequence is sufficient for binding of NucC in vitro. NucC binding to the S. marcescens nuclease promoter P nucA and to the sequence upstream of the P2 late promoter P F is accompanied by DNA bending. NucC protects about 25 nucleotides of the P F upstream region from DNase I digestion, and RNA polymerase protects the promoter region only in the presence of NucC. Template DNA, RNA polymerase holoenzyme, and purified NucC are the only macromolecular components required for transcription from P F in vitro.The NucC protein was originally identified as a positive regulator of extracellular nuclease and bacteriocin 28b production in Serratia marcescens (7,12). NucC is a basic 75-aminoacid protein encoded by what appears to be a cryptic prophage and is a member of the bacteriophage P2 Ogr family of transcriptional activators. These proteins, which are required for late gene expression in P2-and P4-related phages, represent a novel class of prokaryotic zinc finger transcription factors. All of the members of this family are functionally interchangeable, at least to some extent, and have significant amino acid sequence similarity. Four invariant Cys residues, arranged in a Cys-X 2 -Cys-X 22 -Cys-X 4 -Cys motif, are essential for function and involved in the coordination of zinc (8,15,19,21,26). Binding sites for these proteins have been localized upstream of P2 and P4 late promoters and the S. marcescens nuclease promoter (1,10,13,14,33,34); these sites include a conserved element of hyphenated dyad symmetry, TGT-N 12 -ACA. Genetic evidence indicates that members of this family of proteins activate transcription via a specific interaction with the C-terminal domain of the ␣ subunit of RNA polymerase (2,31,35).In vitro analysis of the mechanism of transcription activation by members of the P2 Ogr family has been hampered by the relative insolubility of these proteins. The binding sites in P2 and P4 late promoters were defined by using protein mixtures obtained by copurification with a MalE fusion protein (13,14). The related 186B protein was purified as a Cd 2ϩ derivative that retained partial activity (26). We have succeeded in purifying highly active native NucC protein and report here the analysis of the DNA binding specificity and in vitro activity of purified NucC. MATERIALS AND METHODSBacterial strains and plasmids. The strains and plasmids used in this study are listed in Table 1.Plasmid construction and DNA manipulations. Standard methods wer...

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“…1). Previous deletion analysis of the P2 FETUD operon promoter P F (8) and a more detailed mutational analysis of the P4 P sid promoter (21) implicated this upstream region in promoter function. DNase I protection studies confirmed that this region is bound by P4 Delta, as well as the related activator proteins encoded by phages R73 and PSP3 (9, 10) and a cryptic prophage in Serratia marcescens (17).…”

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Identification of Upstream Sequences Essential for Activation of a Bacteriophage P2 Late Promoter

et al. 2003

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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.P2 belongs to a group of serologically related temperate phages that share the ability to support the lytic multiplication of satellite (helper-dependent) phages P4 and R73. Lytic growth of both P2 and the P2-dependent satellite phages requires the P2 late genes, which encode the proteins involved in capsid and tail synthesis and cell lysis (reviewed in references 4 and 16). P2 late-gene transcription depends upon the P2 ogr gene product. The P4 and R73 ␦ genes encode transcription factors related to P2 Ogr that can directly activate transcription from the P2 late promoters when utilizing P2 as a helper for lytic growth. These activator proteins belong to a family of small zinc binding transcription factors that coordinate one atom of zinc with 4 Cys residues arranged in a conserved CX 2 CX 22 CX 4 C motif (11,15,19). Transcription from two late promoters on the P4 genome, P sid and P LL , is also activated by Ogr or Delta (5, 6).The four P2 late promoters (2, 3) and the two P4 late promoters (5, 6) share features typical of positively regulated bacterial promoters recognized by RNA polymerase carrying the 70 subunit. These promoters lack good homology to the Ϫ35 consensus sequence. Partial homology with the Ϫ10 consensus sequence in the different promoters exists to various extents, with the two most highly conserved nucleotides being retained in all cases. The most striking feature of these six late promoters, as well as other promoters positively regulated by members of this family of transcription factors (7,23,25), is a conserved upstream element of hyphenated dyad symmetry, with the consensus sequence TGT-N 12 -ACA, centered at about position Ϫ55 (Fig. 1). Previous deletion analysis of the P2 FETUD operon promoter P F (8) and a more detailed mutational analysis of the P4 P sid promoter (21) implicated this upstream region in promoter function. DNase I protection studies confirmed that this region is bound by P4 Delta, ...

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Deletion analysis of a bacteriophage P2 late promoter

Cited by 31 publications

References 26 publications

Bacteriophage P2

Bacteriophage P2

Purification and In Vitro Characterization of the Serratia marcescens NucC Protein, a Zinc-Binding Transcription Factor Homologous to P2 Ogr

Identification of Upstream Sequences Essential for Activation of a Bacteriophage P2 Late Promoter

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