Mutations in β′ subunit of Escherichia coli RNA polymerase perturb the activator polymerase functional interaction required for promoter clearance

Transcription activator C employs a unique mechanism to activate mom gene of bacteriophage Mu. The activation process involves, facilitating the recruitment of RNA polymerase (RNAP) by altering the topology of the promoter and enhancing the promoter clearance by reducing the abortive transcription. To understand the basis of this multi-step activation mechanism, we investigated the nature of the physical interaction between C and RNAP during the process. A variety of assays revealed that only DNA-bound C contacts the β′ subunit of RNAP. Consistent to these results, we have also isolated RNAP mutants having mutations in the β′ subunit which were compromised in C-mediated activation. Mutant RNAPs show reduced productive transcription and increased abortive initiation specifically at the C-dependent mom promoter. Positive control (pc) mutants of C, defective in interaction with RNAP, retained the property of recruiting RNAP to the promoter but were unable to enhance promoter clearance. These results strongly suggest that the recruitment of RNAP to the mom promoter does not require physical interaction with C, whereas a contact between the β′ subunit and the activator, and the subsequent allosteric changes in the active site of the enzyme are essential for the enhancement of promoter clearance