Readthrough of transcription terminators can be the result of modification of RNA polymerase (RNAP) to a terminator- resistant form. Nascent transcripts encoded by the two cis- acting antitermination sites (put sites) of bacteriophage HK022 promote such a modification. Immediately after put is transcribed, the elongating RNAP molecule acquires the ability to read through multiple downstream transcription terminators. Put-dependent antitermination has little or no terminator specificity: both Rho-dependent and intrinsic terminators were suppressed. Efficient readthrough depends on the structure of the put transcript (PUT) and a domain located in the beta subunit of RNAP. No additional protein factor is required. This suggests that a direct interaction between nascent PUT and RNAP is a step in antitermination. To probe the PUT-RNAP interaction, we hybridized anti-PUT oligonucleotides to nascent transcripts and measured their effect on terminator readthrough. The oligos were added either at the start of transcription or after the put site had been transcribed but before RNAP had reached the terminator site. To stall RNAP between put and the terminator, we bound Lac repressor between them. We also measured the sensitivity of PUT to RNaseH cleavage after hybridization with the oligos. Our results provide biochemical evidence of a direct PUT:RNAP interaction and support the following working model. PUT consists of 2 stem-loops separated by an unpaired base. Nascent stem-loop 1 promotes the folding of the adjacent stem-loop 2 or its interaction with RNAP, after which stem-loop 1 is dispensable. All or part of stem-loop 2 then binds stably to RNAP and increases its resistance to termination. Since PUT made with T7 RNAP did not promote antitermination when it was added to transcription reactions, we propose that E. coli RNAP must synthesize the PUT it interacts with. This may be because E. coli but not T7 RNAP promotes conversion of PUT to an active form. Our results also suggest that the antitermination defect of a beta mutant of RNAP (Y75N) is a result of its failure to interact with PUT. This and the absence of other Y75N phenotypes suggest that Y75 is part of the PUT interaction domain.Lysogens of phage HK022 are resistant to infection by lambda because the HK022 Nun protein terminates early lambda transcripts. Nun binds to specific sites in nascent lambda RNA and arrests elongating RNAP. We have shown that the nun gene is transcribed by a weak, constitutive prophage promoter, pNUN, located just upstream of the coding sequence. We determined the 5-end of transcripts isolated from lysogens or made in vitro with purified RNAP, and the effect of pNUN mutations on Nun concentration and activity in lysogens. Lysogens contain about several hundred molecules of Nun per cell in stationary phase, and fewer in exponential phase. This small number together with the high efficiency of Nun termination even when the transcripts are abundant suggest that Nun molecules bound to arrested elongation complexes are released in active form after termination and recycle to newly synthesized transcripts. - Transcription Antitermination; Transcription Termination; Bacteriophage HK022; RNA Polymerase; Bacteriophage Lambda
