Electrophoretic Mobility Shift Assay: Analyzing Protein - Nucleic Acid Interactions

IntroductionInteractions between proteins and nucleic acids mediate a wide range of processes within a cell from its cycle to the maintenance of cellular metabolic and physiological balance.These specific interactions are crucial for control of DNA replication and DNA damage repair, regulation of transcription, RNA processing and maturation, nuclear transport, and translation.The characterization of protein-nucleic acid interactions is essential not only for understanding the wide range of cellular processes they are involved in, but also the mechanisms underlying numerous diseases associated with the breakdown of regulatory systems.These include, but are far from being limited to, cell cycle disorders such as cancer and those caused by pathogenic agents that rely on or interfere with host cell machinery.More recently, it has been hypothesized that many neurological disorders such as Alzheimer's, Huntington's, Parkinson's, and polyglutamine tract expansion diseases are a consequence, at least in part, of aberrant protein-DNA interactions that may alter normal patterns of gene expression (Jiménez, 2010).The electrophoretic mobility shift assay (EMSA), also known as gel retardation assay, is a regularly used system to detect protein-nucleic acid interactions.It was originally developed with the aim of quantifying interactions between DNA and proteins (Fried & Crothers, 1981;Garner & Revzin, 1981) and since then evolved to be suitable for different purposes including the detection and quantification of RNA-protein interactions.EMSA is most commonly used for qualitative assays including identification of nucleic acid-binding proteins and of the respective consensus DNA or RNA sequences.Under proper conditions, however, EMSA can also be used for quantitative purposes including the determination of binding affinities, kinetics, and stoichiometry.EMSA is a commonly used method in the characterization of transcription factors, the most intensely studied DNA-binding proteins, and the largest group of proteins in humans, second only to metabolic enzymes.Their purification and identification is crucial in understanding gene regulatory mechanisms.Transcription factors are sequence specific DNA binding proteins that are usually assembled in complexes formed prior to transcription initiation.They bind discreet and specific DNA sequences in the promoter www.intechopen.comGel Electrophoresis -Advanced Techniques 206 region functioning either as an activator or repressor of expression of the targeted gene through protein-protein interactions (reviewed by Simicevic & Deplancke, 2010).Transcription factors play essential roles during development and differentiation.It is well established that disruption of normal function of tissue-specific transcription factors, as a result of mutations, is often associated with a number of diseases including most forms of cancer, neurological, hematological, and inflammatory diseases.Additionally, transcription factors are often found differentially expressed in different pathologies suggesting an at least indirect involvement on the onset or progression of diseases.One of the most prominent examples of the involvement of transcription factors in development and progression of diseases is perhaps the p53 protein.p53 is a transcription factor involved in the modulation of expression of several genes that regulate essential cellular processes such as cell proliferation, apoptosis, and DNA damage repair (reviewed by Puzio-Kuter, 2011).Mutations in p53 that cause loss of function were reported in about 50% of all cancers.It is believed that this loss of function makes cancer cells more prone to the accumulation of mutations in other genes thus facilitating and accelerating the formation of neoplasias (reviewed by Goh et al., 2011).In our laboratory, research is mainly directed to the study of host-pathogen interactions during hepatitis delta virus (HDV) replication and infection.HDV is the smallest human pathogen so far identified and infects human hepatocytes already infected with the hepatitis B virus (HBV).Both viruses have the same envelope proteins that are coded by the HBV DNA genome.HDV is, thus, considered a satellite virus of HBV.The HDV genome consists of a single-stranded, circular, RNA molecule of about 1700 nucleotides.This genome contains only one open reading frame from which two forms of the same protein, the so-called delta antigen, are derived by an editing mechanism catalyzed by cellular adenosine deaminase I.Both forms, small and large delta antigen, were shown to play crucial roles during virus replication: the small delta antigen is necessary for virus RNA accumulation and the large delta antigen plays an important role during envelope assembly (reviewed by Rizzetto, 2009).However, neither protein seems to display any known enzymatic activity.Accordingly, HDV is highly dependent on the host cell machinery for virus replication.It has been shown through EMSA that the small delta antigen binds in vitro to RNA and DNA without any specificity, which is in agreement with one of the roles attributed to the protein as a chaperone (Alves et al., 2010).Making use of different experimental approaches it was possible to identify a number of cellular proteins that interact with HDV antigens or RNA (reviewed by Greco-Stewart & Pelchat, 2010).However, the precise role played by most host factors during the virus life cycle remains elusive.Furthermore, it is highly consensual among HDV researchers that many other cellular factors that interact with delta antigens or HDV RNA remain to be identified and it is crucial to find those that interact with HDV RNA for a better insight on its replication and as possible targets for new therapies.In this chapter we will review the principles of EMSA and its advantages and limitations for the quantitative and qualitative analysis of protein-nucleic acid interactions.The key parameters influencing the quality of protein samples, binding to nucleic acids, complex migration in gels, and sensitivity of detection will be discussed.Finally, an overview of the principles, advantages and disadvantages of methods that are an alternative to gel retardation assays will be provided.www.intechopen.