Mutational analysis of the bacteriophage P2 Ogr protein: truncation of the carboxy terminus

Abstract

The Ogr protein is a 72-residue, zinc-binding transcription factor essential for activation of late gene expression in bacteriophage P2. Analysis of C-terminal truncated proteins generated by stop codon mutagenesis shows that deletion of residues distal to position 51 had negligible effects on Ogr function. More-extensive deletion resulted in unstable products with severely reduced activity. These results, as well as the elfects of other mutations in this region, support the idea that the 21 C-terminal residues are not required for transactivation.The P2 ogr gene encodes a transcription factor essential for activation of the late genes of bacteriophage P2 (for a review, see reference 3). It has been suggested that Ogr binds to promoter regions at a dyad symmetry element centered at position -55 upstream of the operons it controls (10, 25). Furthermore, genetic evidence implies a direct interaction between Ogr and the host RNA polymerase: the rpoA109 allele of the ot subunit of Escherichia coli RNA polymerase specifically imposes a block on P2 late gene transcription which is overcome by suppressor mutations located in the ogr gene (8,24). This suggests that the Ogr protein functions in a transcription initiation complex via specific contacts between Ogr and DNA and also between Ogr and the at subunit of RNA polymerase. Despite considerable efforts, however, no direct experimental evidence for any of the proposed binding interactions of the Ogr protein has yet been obtained, and the mechanism of Ogr action remains elusive.Ogr belongs to a family of functionally and structurally homologous proteins, encoded by P2-and P4-related phages, which show no significant sequence similarity to other known transcription factors. A comparison between Ogr and the related proteins from bacteriophages P4 and 186 and retronphage 4R73 is shown in Figure 1. There is strong conservation of amino acids in the amino-terminal two-thirds of these proteins; seven amino acids, including four cysteines arranged in the pattern CysX2CysX22CysX4Cys, are invariant. This arrangement does not correspond closely to the consensus sequences of any of the known classes of DNA-binding zinc finger proteins (7). While it has been demonstrated that purified Ogr binds zinc (17), the exact role that zinc binding may play in the mode of action of the Ogr protein is still unknown. The carboxy-terminal regions of these proteins show little sequence conservation. They do, however, contain an unusually large number of proline residues and are predicted to be fairly hydrophobic. It has been proposed (16) that this proline-rich region may constitute a transcription activation domain, reminiscent of the proline-rich domain which has been invoked for some eukaryotic transcriptional activators (18).As (14), with reagents, protocols, and bacterial strains supplied in the Phagemid mutagenesis kit (Bio-Rad). Single-stranded, uracil-containing pKG100 template DNA was isolated by infection with the helper phage M13K07 (26). Mutations were identified either by direct se...

The complete nucleotide sequence of φCTX, a cytotoxin‐converting phage of Pseudomonas aeruginosa: implications for phage evolution and horizontal gene transfer via bacteriophages

“…5A). The homology is localized in the N‐terminal portion of the proteins, which has been reported to be essential for the Ogr activity (Gebhardt et al ., 1993; Pountney et al ., 1997). In particular, six amino acid residues including four cysteines that are perfectly conserved in all Ogr proteins are conserved in φCTX ORF34 as well, suggesting that it is an Ogr homologue of φCTX.…”

Metal‐ and DNA‐binding properties and mutational analysis of the transcription activating factor, B, of coliphage 186: A prokaryotic C4 zinc‐finger protein

“…It has been reported that a plasmid-encoded mutant of the P2 Ogr gene product terminating after the first SI amino acids is able to complement a P2 mutant lacking an active Ogr gene in a phage burst assay (Gebhardt et al. 1993).…”

“…Studies chiefly on P2 Ogr and P4 6 indicate that the proteins bind zinc (Lee & Christie, 1990), interact with the a subunit of E. coli RNA-polymerase (Sunshine & Sauer, 1975;Fujiki et al, 1976;Halling et al, 1990;King et al, 1992;Ayers et al, 1994), have a region of the promoter DNA centered at -55 (Julien & Calendar, 1995), and contain a C-terminal region (ca. 20 amino acids) non-essential to protein function (Gebhardt et al, 1993). Further work has been hampered by the insolubility of the purified proteins other than as gene fusion products (Lee & Christie, 1990: Julien & Calendar, 1995.…”

Analysis of DNA Binding by a Eubacterial Zinc Finger Transcription Factor

“…The function(s) of the nonconserved C-terminal residues is unclear. Viable C-terminal deletions of P2 ogr and phage 186 B suggest that this region is dispensable, although it likely contributes to full activity (18,36). Genetic evidence is consistent with a mechanism for transcription activation that involves a direct interaction with the C-terminal domain of the ␣ subunit(s) of RNA polymerase (1,45,50).…”