Transcription activation by the bacteriophage Mu Mor protein requires the C-terminal regions of both alpha and sigma(70) subunits of Escherichia coli RNA polymerase

Middle transcription of bacteriophage Mu requires Escherichia coli RNA polymerase and a Mu-encoded protein, Mor, Consistent with these requirements, the middle promoter, P-m, has a -10 hexamer but lacks a recognizable -35 hexamer, Interactions between Mor and RNA polymerase were studied using in vitro transcription, DNase I footprinting, and the yeast interaction trap system, We observed reduced promoter activity in vitro using reconstituted RNA polymerases with C-terminal deletions in alpha or sigma(70). As predicted if alpha were binding to P-m, we detected a polymerase-dependent footprint in the -60 region, Reconstituted RNA polymerases containing Ala substitutions in the alpha C-terminal domain were used to assay Mor-dependent transcription from P-m in vitro. The D258A substitution and alpha deletion gave large reductions in activation, whereas the L262A, R265A, and N268A substitutions caused smaller reductions, The interaction trap assay revealed weak interactions between Mor and both alpha and sigma(70); consistent with a key role of alpha-D258, the D258A substitution abolished interaction, whereas the R265A substitution did not, We propose that: (i) alpha-D258 is a Mor ''contact site''; and (ii) residues Leu-262, Arg-265, and Asn-268 indirectly affect Mor-polymerase interaction by stabilizing the ternary complex via alpha-DNA contact.