Mutations affecting two adjacent amino acid residues in the alpha subunit of RNA polymerase block transcriptional activation by the bacteriophage P2 Ogr protein

Abstract

The bacteriophage P2 ogr gene product is a positive regulator of transcription from P2 late promoters. The ogr gene was originally defined by compensatory mutations that overcame the block to P2 growth imposed by a host mutation, rpoA109, in the gene encoding the a subunit of RNA polymerase. DNA sequence analysis has confirmed that this mutation affects the C-terminal region of the a subunit, changing a leucine residue at position 290 to a histidine (rpoAL290H). We have employed a reporter plasmid system to screen other, previously described, rpoA mutants for effects on activation of a P2 late promoter and have identified a second allele, rpoA155, that blocks P2 late transcription. This mutation lies just upstream of rpoAL290H, changing the leucine residue at position 289 to a phenylalanine (rpoAL289F). The effect of the rpoAL289F mutation is not suppressed by the rpoAL29OH-compensatory P2 ogr mutation. P2 ogr mutants that overcome the block imposed by rpoAL289F were isolated and characterized. Our results are consistent with a direct interaction between Ogr and the a subunit of RNA polymerase and support a model in which transcription factor contact sites within the C terminus of a are discrete and tightly clustered.Many eubacterial genes and operons are under positive control, and the structures and DNA-binding sites of a number of prokaryotic transcriptional activators have been well characterized (for a review, see reference 1). The precise mechanism by which these regulatory proteins catalyze the initiation of transcription remains largely obscure. The identification of lambda repressor (9, 24, 27, 28) and catabolite gene activator protein (CAP) (3, 15, 65) mutants that were defective as activators but retained DNA-binding ability suggested that these proteins function by interacting directly with RNA polymerase. A rapidly growing body of genetic evidence supports a functional interaction between the C-terminal region of the ax subunit(s) of DNA-dependent RNA polymerase and specific transcriptional activators.The earliest indication that the a subunit plays a role in positive control was provided by Escherichia coli mutation rpoA109 (61), which prevents lytic growth of phage P2 and satellite phage P4 by interfering with phage late-gene expression. This mutation results in a leucine-to-histidine change in the C-terminal region of the ax subunit (16 general pattern that has emerged from the characterization of these mutants is that they are altered in the C-terminal region of the a polypeptide and that each rpoA mutation appears to be fairly specific, affecting only one or a small group of positively regulated promoters. Those mutations that affect the responsiveness of RNA polymerase to each activator tend to be tightly clustered, suggesting that each transcriptional activator interacts with a discrete domain within the C-terminal region of the a subunit to stimulate transcription.To further define a domain of the ao subunit involved in interaction with the activators of P2 late-gene expression, we have dete...

Transcription of theSalmonellaInvasion Gene Activator,hilA, Requires HilD Activation in the Absence of Negative Regulators

“…5A and B) (18,25). Residues in the third alpha helix, including leucine 289, have been shown to be important for activation of the P2 late promoter, and allele-specific suppressor mutations have been identified in the Ogr activator that overcome rpoA mutations to allow P2 latepromoter expression (3). Therefore, we propose that leucine 289 is important for a favorable interaction between the ␣CTD and HilD to occur, activating transcription of hilA (Fig.…”

Bacteriophage P2

“…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. 73 Unlike ogr, the homologous B gene in 186 is under direct control of the phage immunity repressor.…”

Molecular Characterization of the Growth Phase-Dependent Expression of the lsrA Gene, Encoding Levansucrase of Rahnella aquatilis

“…The se-quence of binding site I, centered at Ϫ55, is an exact match with the consensus binding site sequence of the P2 Ogr family proteins. The P2 Ogr protein activates the transcription of the target gene through direct interaction with the ␣ subunit of the RNA polymerase complex (2,12). The proximity of binding site I to the promoter corresponds to the predicted model for action of the activators.…”