Analysis of polymerase II elongation complexes by native gel electrophoresis. Evidence for a novel carboxyl-terminal domain-mediated termination mechanism.
Genetic and proteomic approaches have identified numerous proteins that are potentially involved in regulating transcriptional elongation, but the mechanisms of action of these proteins remain largely unknown. We describe an experimental approach using native gel electrophoresis for studying interactions of elongation factors with isolated Pol II elongation complexes. The gel distinguishes Pol IIA and Pol IIB containing complexes. The interaction of DSIF (Spt4/Spt5) with the elongation complexes can be readily detected, and this association is not dependent on the carboxyl-terminal domain of the largest subunit of Pol II. We also report the surprising observation that a monoclonal antibody that binds the carboxyl-terminal domain of Pol II triggers the dissociation of the elongation complex. The action of the antibody could be mimicking the action of cellular factors involved in transcription termination.
Zhang
Zhiqiang
Z
Center for Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Wu
Chwen-Huey
CH
Gilmour
David S
DS
D000818:Animals; D004330:Drosophila; D004587:Electrophoresis, Agar Gel; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012319:RNA Polymerase II / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D021122:Protein Subunits; D012319:RNA Polymerase II