Related Experiment Video
Updated: May 27, 2026
12:44
Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example
Published on: December 3, 2014
Probing endogenous RNA polymerase II pre-initiation complexes by electrophoretic mobility shift assay
Emmanuelle Wilhelm1, Christopher Takacs, Brendan Bell
1RNA Group, Département de microbiologie et d'infectiologie, Faculté de médecine et sciences de la santé, Université de Sherbrooke, Sherbrooke, QC, Canada.
Methods in Molecular Biology (Clifton, N.J.)
|November 25, 2011
Summary
Researchers developed a new method to study how RNA polymerase II (Pol II) and its associated factors bind to DNA. This technique, optimized electrophoretic mobility shift assays (EMSAs), allows for the analysis of endogenous Pol II pre-initiation complexes (PICs).
Area of Science:
- Molecular Biology
- Gene Regulation
- Biochemistry
Background:
- RNA polymerase II (Pol II) is essential for transcribing protein-coding genes, microRNAs, and small nuclear RNAs in eukaryotes.
- Pol II recruitment to DNA promoters involves general transcription factors (GTFs), forming pre-initiation complexes (PICs).
- Existing methods using purified GTFs cannot fully explain the diversity observed in core promoters and PIC composition.
Purpose of the Study:
- To develop a technique for analyzing the DNA binding of endogenous PICs, which has been technically challenging.
- To identify the complete set of factors responsible for PIC specificity.
- To overcome hurdles related to PIC size, dynamic interactions, and DNA topology.
Main Methods:
- Optimization of electrophoretic mobility shift assays (EMSAs).
- Analysis of endogenous Pol II PICs from nuclear extracts of human cells.
- Development of a robust and sensitive EMSA method.
Main Results:
- Successful detection of endogenous Pol II PICs using the optimized EMSA.
- Establishment of a sensitive method for analyzing endogenous PIC formation.
- Demonstration of a viable approach to study PICs directly from cellular extracts.
Conclusions:
- The optimized EMSA method enables the study of endogenous Pol II PICs, addressing limitations of previous techniques.
- This advancement is crucial for understanding the full complement of factors involved in transcription initiation.
- The technique provides a new tool for investigating the diversity of PICs and core promoter interactions.
