Characterization of the effects of Escherichia coli replication terminator protein (Tus) on transcription reveals dynamic nature of the tus block to transcription complex progression.
We have characterized the blocks to progression of T7 and T3 RNA polymerase transcription complexes created when a Tus protein is bound to the template. The encounter with Tus impedes the progress of the transcription complexes of either enzyme. The duration of the block depends on which polymerase is used and the orientation of Tus on the DNA. Both genuine termination (dissociation of the transcription complex) and halting followed by continued progression after the block is abrogated are observed. The fraction of complexes that terminates depends on which polymerase is used and on the orientation of the Tus molecule. The efficiency of the block to transcription increases as the Tus concentration is increased, even if the concentration of Tus is already many times in excess of what is required to saturate its binding sites on the template in the absence of transcription. The block to transcription is rapidly abrogated if an excess of a DNA containing a binding site for Tus is added to a transcription reaction in which Tus and template have been preincubated. Finally, we find that transcription will rapidly displace Tus from a template under conditions that generate persistent blocks to transcription. These observations reveal that during the encounter with the transcription complex Tus rapidly dissociates from the template but that at sufficiently high concentrations Tus usually rebinds before the transcription complex can move forward. The advantage of a mechanism which can create a persistent block to transcription or replication complex progression, which can nevertheless be rapidly abrogated in response to down regulation of the blocking protein, is suggested.
Guajardo
R
R
Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7760, USA.
Sousa
R
R
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C061910:tus protein, Bacteria; C489236:tus protein, E coli; D012321:DNA-Directed RNA Polymerases