Molecular Basis Of Conversion Of Transcription Termination Factor Nusa Into An Antiterminator

The well conserved bacterial transcription elongation/termination factor NusA is an essential component of the N‐mediated transcription antitermination machinery of the lambdoid phages. N‐NusA interaction during the transcription elongation process is mandatory for N to function. Mode of this interaction holds the key to understand the mechanism of conversion of NusA into an antiterminator by N. We have shownthat upon binding to the EC, the interaction surface of NusA for N changes from the NusA‐AR1 region to its NTD domain. We proposed that the close proximity of this N‐interaction site of NusA to its RNAP‐binding surface (β‐flap) enables N to exert conformational changes to RNAP allosterically via NusA. We further hypothesized the following. In the presence of N, i) NusA bind to a new site in the transcription elongation complex (EC) and/or, ii) NusA changes the conformations of the flap‐domain.In vitro competition assays revealed that the association of NusA to the EC stabilized several fold in the presence of N, which is indicative of an altered NusA‐EC interaction pattern. Two mutants, G1045D and R1058L, in the flap domain were known to affect N and Q‐mediated antitermination, respectively. These mutants unlike RNA‐exit channel mutants (P251S, P254L, G336S, and R270S; likely to be part of the N‐CTD interaction sites), were not suppressed by a suppressor, L108F, present in the RNAP‐binding domain of N. They were not‐defective for N‐mediated antitermination at the Rho‐dependent terminators, where N‐NusA‐RNAP interactions are less important. Finally, these two mutants exhibited severe synthetic defects with a NusA mutant, V8A, partially impaired for N‐binding. These results indicate that the antitermination defects of these β‐flap mutants were due to incomplete transition of flap domain by NusA in the presence of N, and thereby supported the aforementioned hypothesis.