Evidence that the KH RNA-binding Domains Influence the Action of the E. coli NusA Protein

The NusA transcription elongation protein, which binds RNA, contains sequences corresponding to the S1 and KH classes of identified RNA binding domains. An essential function in E. coli, NusA is also one of the host factors required for action of the N transcription antitermination protein of λ. Tandem KH domains have been identified downstream of the S1 domain. We changed the first Gly to Asp of the GXXG motif, a tetrapeptide diagnostic of KH domains, of both NusA KH domains. The change in the first, G253D, has a large effect, while the change in the second, G319D, has a small effect on NusA action. The changes in both KH domains interfere with NusA binding to RNA. A change of a highly conserved Arg in the S1 domain, R199A, has previously been reported to interfere with RNA binding while exerting a small effect on NusA action. However, a nusA allele with both the R199A and G319D changes encodes a functionally inactive NusA protein. These studies provide direct evidence that the both KH as well as the S1 RNA binding domains are important for NusA action in support of bacterial viability as well as transcription antitermination mediated by the λ N protein.