Analysis of factor interactions with RNA polymerase II elongation complexes using a new electrophoretic mobility shift assay.
The elongation phase of transcription by RNA polymerase II (RNAP II) is controlled by a carefully orchestrated series of interactions with both negative and positive factors. However, due to the limitations of current methods and techniques, not much is known about whether and how these proteins physically associate with the engaged polymerases. To gain insight into the detailed mechanisms involved, we established an experimental system for analyzing direct factor interactions to RNAP II elongation complexes on native gels, namely elongation complex electrophoretic mobility shift assay (EC-EMSA). This new assay effectively allowed detection of interactions of TFIIF, TTF2, TFIIS, DSIF and P-TEFb with elongation complexes generated from a natural promoter using an immobilized template. As an application of this assay system, we characterized the association of transcription elongation factor DSIF with RNAP II elongation complexes and discovered that the nascent transcript facilitated recruitment of DSIF. Examples of how the system can be manipulated to address different questions are provided. EC-EMSA should be useful for further investigation of factor interactions with RNAP II elongation complexes.
Cheng
Bo
B
Molecular and Cellular Biology Program and Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA.
Price
David H
DH
D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D042925:Positive Transcriptional Elongation Factor B / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000378:metabolism*
D009687:Nuclear Proteins; D012333:RNA, Messenger; C112224:SUPT5H protein, human; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; D042925:Positive Transcriptional Elongation Factor B; D012319:RNA Polymerase II