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 autogenously 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 reproduced in a purified transcription system but requires an additional factor, NusA. Our earlier in vitro studies showed that NusA is required for RNA polymerase pausing at the termination site; such paused complexes are further stabilized by L4, which presumably accounts for L4's stimulation of termination in vivo. Here we show that NusA is not absolutely required for RNA polymerase to recognize the attenuation site: at low (5 mu M) UTP concentration, RNA polymerase pauses at the site, although the paused transcription complex formed in the absence of NusA can be further stabilized by subsequent addition of the protein. Furthermore, RNA polymerase pausing at the attenuation site is not sufficient for the L4 effect, since L4 cannot stabilize a transcription complex 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 the notion that L4 is a stable component of a paused transcription complex.