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 α or σ70. As predicted if α were binding to P(m), we detected a polymerase-dependent footprint in the - 60 region. Reconstituted RNA polymerases containing Ala substitutions in the α C-terminal domain were used to assay Mor-dependent transcription from P(m) in vitro. The D258A substitution and α 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 α and σ70; consistent with a key role of α-D258, the D258A substitution abolished interaction, whereas the R265A substitution did not. We propose that: (i) α-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 α-DNA contact.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology