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Nucleic Acids Research Advance Access published online on October 1, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn630
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

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

Bo Cheng1 and David H. Price1,2,*

1Molecular and Cellular Biology Program and 2Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA

*To whom correspondence should be addressed. Tel: +1 319 335 7910; Fax: 319 335 9570; Email: david-price{at}uiowa.edu

Received August 7, 2008. Revised September 9, 2008. Accepted September 12, 2008.

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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