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Analysis of factor interactions with RNA polymerase II elongation complexes using a new electrophoretic mobility shift assay
Journal article   Open access   Peer reviewed

Analysis of factor interactions with RNA polymerase II elongation complexes using a new electrophoretic mobility shift assay

Bo Cheng and David H Price
Nucleic acids research, Vol.36(20), pp.e135-e135
11/2008
DOI: 10.1093/nar/gkn630
PMCID: PMC2582608
PMID: 18832375
url
https://doi.org/10.1093/nar/gkn630View
Published (Version of record) Open Access

Abstract

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.
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