Netropsin specifically enhances RNA polymerase II termination at terminator sites in vitro.
We describe an in vitro system that emulates the specific and efficient transcriptional termination associated with the human gastrin gene terminator in vivo. The system involves a dC-tailed DNA template containing the gastrin gene terminator sequence, purified RNA polymerase II, and purified elongation factor TFIIS. In this system, the basal level of termination by RNA polymerase II at the gastrin gene terminator is specifically enhanced by netropsin, an (A + T)-rich minor groove-binding peptide. This enhanced termination is maintained even with TFIIS, which normally suppresses termination at this site. In vitro termination is terminator sequence-specific. Mutant sequences that reduce or abolish termination in vivo show corresponding reductions in activity in the in vitro system. This in vitro emulation of in vivo activities of wild-type and mutant terminators strongly suggests that netropsin and a putative termination factor may share some aspects of their biochemical mechanisms. The general applicability of this system to the study of RNA polymerase II elongation and termination is suggested by the enhancement of termination seen at both the gastrin and human histone H3.3 gene terminators.
Ueno
A
A
Department of Biochemistry and Molecular Biology, University of Chicago, IL 60637.
Baek
K
K
Jeon
C
C
Agarwal
K
K
D001483:Base Sequence; D005755:Gastrins / Q000235:genetics; D006657:Histones / Q000235:genetics; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009429:Netropsin / Q000494:pharmacology*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012319:RNA Polymerase II / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013728:Terminator Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*
D005755:Gastrins; D006657:Histones; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors; D009429:Netropsin; D012319:RNA Polymerase II