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

Abstract

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 α subunits of the host RNA polymerase. An alanine scan spanning the putative activation target in the α C-terminal domain (αCTD) 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 αCTD 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, define a surface-exposed patch on the αCTD important for Ogr-dependent activation. These residues, adjacent to the recently identified DNA-binding determinants, constitute a newly identified 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-α contacts, in addition to α-Ogr contacts, may be important in P2 late transcription. Other residues implicated in the interaction of α with CAP are not required for activation by Ogr, consistent with previous genetic evidence suggesting that these activators contact different sites on the αCTD.