Mutational changes of conserved residues in the Q-loop region of transcription factor Rho greatly reduce secondary site RNA-binding

Transcription factor Rho of Eschericia coli is a ring-shaped homohexameric protein that terminates transcripts by its action on nascent RNAs. To test the functional importance of the phylogenetically highly conserved residues of the Q-loop region, four mutant Rho proteins, S281A, K283A, T286A and D290A, were isolated and analyzed for their biochemical properties. All four proteins were very defective in terminating transcripts in vitro at the bacteriophage λ tR1 terminator and had corresponding defects in ATP hydrolysis activated by λ cro RNA. Although the four proteins were normal or near normal in their sensitivity to cleavage with H2O2 in the presence of Fe-EDTA and in their ability to bind to λ cro RNA and ATP, they were defective in RNA-specific, secondary site interactions. This was indicated by the lack of protection from cleavage at their Q-loops by oligo(C) in the presence of poly(dC), and their defects in ATP hydrolysis activated by oligo(C) in the presence of poly(dC). This evidence, together with the observations that cleavage of the Q-loop residues is protected specifically by RNA, suggests that the Q-loop makes interactions with RNA that are essential for activation of ATP hydrolysis and the termination of transcription.