The Nun protein of the lambdoid phage HK022 blocks lambda growth by terminating transcription at (or near) the lambda nut sites. An HK022 lysogen carrying a fusion of the lambda pR promoter and nutR site to a gal operon that lacks its own promoter is, therefore, Gal-. To characterize the target of Nun action, spontaneous Gal+ revertants of this strain were isolated and characterized. Two cis-acting mutations are located in the fusion and represent transversions of conserved nucleotides within the boxA sequence (CGCTCTTA) of nutR. One mutation, (CTCTCTTA), is identical with boxA5. The second, boxA16 (CGCTATTA), has not been reported previously. In the absence of Nun, both boxA mutants reduce gal expression. Analysis of in vivo fusion RNA indicates that the mutations increase termination at or near tR1, a rho-dependent lambda terminator located upstream from the fusion point. In contrast to the nutR+ fusion, Nun stimulates gal expression in the boxA mutants by suppressing transcription termination in the tR1 region. Nun antitermination, however, does not extend to distal terminators. The lambda N-function also suppresses termination at or near tR1 in the mutant fusions. N fails to suppress terminators distal to tR1 in the boxA5 fusion, but displays persistent antitermination activity in the boxA16 fusion. A similar reversal of Nun activity occurs when wild-type fusions are introduced into nusA1, nusB5 or nusE71 hosts. We therefore suggest that Nun and N can interact with RNA polymerase in the absence of wild-type boxA, nusA, nusB or nusE, but that the complex formed with mutant components differs functionally from wild-type.
Lambda nutR mutations convert HK022 Nun protein from a transcription termination factor to a suppressor of termination
ROBLEDO, RENATO;
1990-01-01
Abstract
The Nun protein of the lambdoid phage HK022 blocks lambda growth by terminating transcription at (or near) the lambda nut sites. An HK022 lysogen carrying a fusion of the lambda pR promoter and nutR site to a gal operon that lacks its own promoter is, therefore, Gal-. To characterize the target of Nun action, spontaneous Gal+ revertants of this strain were isolated and characterized. Two cis-acting mutations are located in the fusion and represent transversions of conserved nucleotides within the boxA sequence (CGCTCTTA) of nutR. One mutation, (CTCTCTTA), is identical with boxA5. The second, boxA16 (CGCTATTA), has not been reported previously. In the absence of Nun, both boxA mutants reduce gal expression. Analysis of in vivo fusion RNA indicates that the mutations increase termination at or near tR1, a rho-dependent lambda terminator located upstream from the fusion point. In contrast to the nutR+ fusion, Nun stimulates gal expression in the boxA mutants by suppressing transcription termination in the tR1 region. Nun antitermination, however, does not extend to distal terminators. The lambda N-function also suppresses termination at or near tR1 in the mutant fusions. N fails to suppress terminators distal to tR1 in the boxA5 fusion, but displays persistent antitermination activity in the boxA16 fusion. A similar reversal of Nun activity occurs when wild-type fusions are introduced into nusA1, nusB5 or nusE71 hosts. We therefore suggest that Nun and N can interact with RNA polymerase in the absence of wild-type boxA, nusA, nusB or nusE, but that the complex formed with mutant components differs functionally from wild-type.
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