ROLE OF NUSA IN L4-MEDIATED ATTENUATION CONTROL OF THE S10 R-PROTEIN OPERON OF ESCHERICHIA-COLI

The transcription of the 11 gene S10 operon of Escherichia coli is aut ogenously regulated by one of the operon's products, ribosomal protein L4. This protein stimulates termination of transcription in vivo at a specific site within the S10 leader. The in vivo effect can be reprod uced in a purified transcription system but requires an additional fac tor, NusA. Our earlier ill vitro studies showed that NusA is required for RNA polymerase pausing at the termination site; such paused comple xes are further stabilized by L4, which presumably accounts for L4's s timulation of termination in vivo. Here we show that NusA is not absol utely required for RNA polymerase to recognize the attenuation site: a t low (5 mu M) UTP concentration, RNA polymerase pauses at the site, a lthough the paused transcription complex formed in the absence of NusA can be further stabilized by subsequent addition of the protein. Furt hermore, RNA polymerase pausing at the attenuation site is not suffici ent for the L4 effect, since L4 cannot stabilize a transcription compl ex paused at the attenuation site in the absence of NusA. We have been able to isolate paused complexes formed without NusA and/or L4; such complexes are active upon re-addition of NTPs, and respond as expected to the addition of L4 or NusA. Our experiments are consistent with th e notion that L4 is a stable component of a paused transcription compl ex.