Abstract
We studied release of trp leader RNA and trp template DNA from RNA polymerase during transcription termination at the attenuator of the trp operon of Escherichia coli. Preliminary evidence had suggested that a stable ternary complex was formed at the trp attentuator. We observed that the complexes between RNA polymerase and trp leader RNA and the DNA template produced during transcription were labile at high salt concentrations and were undetectable when transcription was performed in the presence of heparin. These characteristics are atypical of the stable transcription termination complexes described by others (Richardson, J. P., and Conaway, R. (1980) Biochemistry 19, 4293-4299; Shigesada, K., and Wu, C. (1980) Nucleic Acids Res. 8, 3355-3369). We successfully reconstituted polymerase-trp leader RNA complexes in simple mixing experiments; these and other studies indicated that it is core polymerase that binds the leader transcript and the DNA template. In agreement with this conclusion, it was observed that sigma factor inhibited binding of RNA polymerase to the trp leader transcript and the DNA template and displaced leader RNA from RNA polymerase during transcription. It seems likely that small amounts of core polymerase present in the holoenzyme preparation, or generated during transcription, are responsible for the nonspecific binding of RNA transcript and DNA template. Our findings, therefore, suggest that the transcription termination event at the trp attenuator normally involves spontaneous dissociation of polymerase, template, and RNA transcript.
Highlights
We studied release of trp leader RNA and trp tem- the transcribingpolymerase moleculesterminate transcription plate DNA from RNA polymerase during transcription at the attenuator, producing a transcript 140 nucleotides in termination at the attenuator of the trp operon of Esch- length [3]
We observed that the complexes between RNA polymerase and trp leader RNA and the DNA template produced during transcription were labile at high salt concentrations and were undetectable when transcription was performed in the presence of heparin
We successfully reconstituted polymerase-trp leader RNA complexes in simple mixing experiments; these and other studies indicated that attenuator continue to the eonfdthe fragment and synthesize a read-through transcript (Fig. 1).Previous studiessuggested that the leader transcript and DNA template remained stably associated with RNA polymerase in a ternary termination complex that formed at the attenuator [4].’To examine this conclusion, transcription complexes produced in vitrowere analyzed by measuring retention of the leader transcript and DNA template on nitrocellulose filters
Summary
To examine this possibility we compared retention of nascent leader RNAin a transcription reactionto. Complex formation between RNA polymerase, the leader that of purified leader RNA addedto a transcription reaction transcript, andthe DNA template was analyzed by measuring retention of labeled RNA and/or DNA on nitrocellulose fil- 'Underthe conditions we generally used in in vitro reactions ters. When reaction mixtures were analyzed folloawtyinpgical approximately 3 to 10%of the template molecules were transcribed.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
