Interactions of an Arg-rich region of transcription elongation protein NusA with NUT RNA: Implications for the order of assembly of the lambda N antitermination complex in vivo

The E. coli NusA transcription elongation protein (NusA(Ec)), identified be cause of its requirement for transcription antitermination by the N protein , has an Arg-rich S1 RNA-binding domain. A complex of N and NusA with other host factors binding at NUT sites in the RNA renders RNA polymerase termin ation-resistant. An E. coli haploid for nusA944, having nine different codo ns replacing four normally found in the Arg-rich region, is defective in su pport of N action. Another variant, haploid for the nusAR199A allele, with a change in a highly conserved Arg codon in the S1 domain, effectively supp orts N-mediated antitermination. However, nusAR199A is recessive to nusA944 , while nusA(Ec) is dominant to nusA944 for support of N-mediated antitermi nation, suggesting a competition between NusA944 and NusAR199A during compl ex formation. Complex formation with the variant NusA proteins was assessed by mobility gel shifts. NusAR199A, unlike NusA,, and NusA944, fails to for m a complex with N and NUT RNA. However, while NusAR199A, like wild-type Nu sA, forms an enlarged complex with NUT RNA, N, RNA polymerase, and other ho st proteins required for efficient N-mediated antitermination, NusA944 does not form this enlarged complex. Consistent with the in vitro results, NusA 944 prevents NusAR199A but not NusA(Ec) from forming the enlarged complex. The simplest conclusion from these dominance studies is that in the formati on of the complete active antitermination complex in vivo, NusA and N bindi ng to the newly synthesized NUT RNA precedes addition of the other factors. Alternative less effective routes to the active complex that allows bypass of this preferred pathway may also exist. (C) 2001 Academic Press.