ELECTROPHORETIC MOBILITY SHIFT ASSAY: A Method for Analysing Protein-DNA Interactions

Introduction

Biological processes such as transcription, translation, and DNA repair all depend on the specific interactions between proteins and DNA.  These DNA-protein interactions are necessary for the growth, development and survival of organisms in the three domains of life, and perturbations that affect these complex interactions account for the numerous diseases, including cancers in humans [1, 2].  These DNA-binding proteins may function as structural proteins, enzymes, transcription factors and co-factors [2]. Owing to these invaluable roles, It is therefore of utmost importance that we study the mechanisms by which DNA-binding proteins specifically interact with their target DNA (and RNA) to bring about changes in gene expressions or regulation or cellular events [2].

Several techniques have been developed for the study of DNA-protein interactions, which have helped in the elucidation of the mechanisms of complex formation between DNAs and proteins.  These techniques include in vitro methods such as electrophoretic mobility shift assay (EMSA), footprinting assays, phage-display and proximity ligation assay; and in vivomethods such as DNA adenine methyltransferase identification and chromatin immunoprecipitation, as well as in silico tools [2].  The various approaches above have their advantages and drawbacks, and are designed to assay specific interaction parameters [2].

This essay will elaborate on the EMSA approach: describe the general principles including the methods, factors affecting resolution of complex, applications, limitations and draw reasonable conclusions.

EMSA: Definition and Basic Principles

This technique, also called Gel Retardation Assay, is a rapid, simple, efficient and highly sensitive method used for the study of DNA-protein interactions [5].  EMSA is widely used for the identification of DNA-binding proteins (qualitative use), and to determine the binding affinities, stoichiometry, kinetics (quantitative use), and conformational changes of the interactions between DNAs and proteins [1, 5]. It has been particularly useful in the study of how transcription factors bind – either as repressors or activators – to specific promoter regions in DNA to regulate gene expression [1].  This in vitro technique relies on the principle that the complex formed between protein and its target site on DNA will migrate slowly towards the positive bottom end in a gel matrix compared to unbound (or free) DNA, thereby causing a shift that can be easily detected after running the samples on a polyacrylamide or agarose gel [2, 5]. The popularity and versatility of EMSA amongst researchers is attributed to its numerous unique features when compared to other methods. These features are explained below:

• High sensitivity: The technique is very sensitive since low concentrations (0.1mM) and sample volumes (20 uL) can be used to give detectable bands when analysed by autoradiography or with ethidium bromide [1].
• Wide range of samples: A variety of nucleic acids (in size and structures) and proteins (including heavy complexes) can be used to assay the complex interactions [1].
• Again, there is the choice of using either crude protein extracts or purified recombinant proteins. The latter is very useful when the researcher is interested in characterising DNA-binding proteins contained in a nuclear cell extract [1].

Methods Involved in EMSA

Five basic steps are usually carried out in a conventional EMSA protocol [1, 4].  An overview of the steps is sequentially listed below.

• Preparation of purified or crude protein sample, and
• Preparation of nucleic acid: either radioactively labelled (usually with ³²P or occasionally with biotin, fluorophores or digoxigenin) or unlabelled. The sources of DNA could be from cloned DNA (50-400bp) or synthetic oligonucleotide (20-70 nucleotides; [2]).
• Binding reactions: considerations are made for buffer conditions and use of additives to reduce non-specific binding of protein to DNA sequence [1]. For example, studies on the human recombinase rad51 and the E. coli CAP protein show that they require ATP and cAMP, respectively for efficient binding to DNA [6, 7].
• Non-denaturing gel electrophoresis to separate free nucleic acid from preformed complex.
• Lastly, detection of the outcome, usually with ethidium bromide for unlabelled DNA or by autoradiography for labelled DNA. These steps are as depicted in Fig. 1 below.

Factors Affecting Resolution of Complex

The inherent properties of the protein-nucleic acid complex act to affect their relative mobility (R_L, defined as observed distance migrated divided by expected distance) through the gel and the resolution of the complex [8].  These properties include the ratio of the relative mass of the two components that form the complex, charge alteration, and the conformational changes in DNA upon protein binding [8].  Other factors such as gel matrix compositions and concentrations, incubation temperature are external factors that also affect the resolution of the complex [8].  In addition, non-specific DNA-binding proteins (in crude cell extracts) also affect the mobility and hence, an EMSA result outcome [1].  To circumvent the effect of non-specific DNA-binding proteins, non-specific DNAs such as salmon sperm, synthetic poly(dI:dC) are included in the assay so that the non-specific proteins can rather bind to the sites on them [5].  Two of the above factors are explained further in the following paragraph. As would be expected, the relative mobility of the protein-DNA complex decreases as the protein size increases as has been studied for the transcriptional regulator, GCN4 but an increase in the length of DNA exhibits the opposite effect [8].  The latter scenario can be observed when Lac repressor binds to operator DNA (of between 80-500 bp).  In this case, the R_L value rises from 0.5 to 0.68 [9].   It can therefore be inferred that it is the individual mass of the protein and DNA fragment that determines the R_L rather than the absolute mass of the complex.  Moreover, concentrations and compositions of gels also influence the results obtained from an EMSA [8].  A tiny pore diameter and one that allows the migration of both free and bound DNA complex across the gel is chosen.  Polyacrylamide gel pore size averages about 50-200 A⁰ for an acrylamide concentration of between 4 and 10%; and this causes an intense frictional drag on the complex as they migrate across the gel [8].  On the other hand, the pore diameters of agarose gels are wider compared to those of polyacrylamide gels and are therefore, only suitable to resolve large protein masses [8].  Despite these differences in pore sizes, the two gel types are used to resolve complexes.

EMSA Applications

In addition to its use for the study of the associations between nucleic acids and proteins, the technique is also useful in the study of parameters such as binding constants and conformational changes in the DNA molecules brought about by protein binding [1].

1. Study of conformational changes in DNA: The interactions between proteins and its cognate sites on DNA may result in bending of the DNA molecule.  This alteration in structure leads to a reduced relative mobility; and the degree of curvature is dependent on the binding angle and the position of the bend relative to the ends of the DNA [8]. A lower relative mobility is more pronounced if the bend is in the middle of the DNA fragment [8].  Variants of the EMSA method such as circular permutation assay (identifies presence of a bend, see fig. 2 below), and helical phase assay (determines bend direction) have been developed to study the effects of protein-induced bending on DNA [10]. The formal variant, for example, has been used to show that proteins such as TFIIIA, c-AMP activating protein (CAP) and TATA binding protein (TBP) induce bends in DNA [3, 11].  Hence, EMSA method can be used to identify which proteins induce bending in DNA upon binding.
2. Binding Constants: EMSA can also be used to measure the kinetic and thermodynamic parameters of the interactions between proteins and DNAs.  Consider the chemical reaction [5]:If a relatively strong interaction occurs between DNA and protein, then Ka > Kd, and therefore, two bands are observed: complex PD and free DNA (fig. 3). In contrast, weak interactions means that Kd > Ka, and therefore, a fainter band (corresponding to complex PD) and a more intense smear are produced [5].  Although the interactions in the second scenario are weak, complex stability is maintained through molecular sequestration and by ‘’cage effect’’ (prevents dissociated proteins from escaping; [5]).  The binding constant can then be extrapolated by measuring the amount of complex formed relative to protein concentration at equilibrium [1].  An example is illustrated in fig. 3 below for the association of small delta protein with DNA [1].
3. Monitoring protein-DNA complex formation from crude cell extracts: One of the many advantages of EMSA is that both purified proteins and crude cell extract proteins can be used to bind DNA and monitor complex formation [2]. Although there is the problem of non-specific DNA binding when crude cell extract is used, this can be easily overcome by using shorter length of DNA fragment (to limit the binding sites) or by using an EMSA variant called supershift assay in which target-specific antibodies are used to reduce the relative mobility of the complex in gel [5]. Hence, a secondary mobility shift is produced with bound DNA and protein of interest. On the other hand, purified recombinant proteins give accurate and useful data. Parameters such as binding constant and binding affinity and effects of factors such as divalent metals can be easily obtained [5].

   Despite its numerous applications, EMSA is faced with some challenges. First, the complex formed are not always at chemical equilibrium when run on a gel which could result into wrong interpretation of, for example, the molecular weight of the complex formed [5]. Second, EMSA results provide little information about where the protein specifically binds to on the DNA sequence. This information can be readily obtained from the chemical footprinting assay [1, 5, 8]. However, deletion mutations of the DNA fragment have been reported that identifies the binding sequence using EMSA technique [12]. Finally, kinetic studies are limited due to the significant amount of time needed to prepare the sample mix, and then run it on the gel [1].

Conclusion

Electrophoretic mobility shift assay has yielded useful information on the mechanisms by which certain proteins specifically bind to DNA sequences and whether or not these interactions induce any conformational changes in DNA.  The technique has also allowed for the study of quantitative parameters such as the kinetics of the interactions between DNAs and proteins; and to identify which proteins bind to a particular DNA sequence from a crude cell extract.  Although of importance, EMSA is faced with some limitations and further work to overcome these would be valuable in molecular biology studies.

References

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11. Wu, H.-M. and D.M. Crothers, The locus of sequence-directed and protein-induced DNA bending. Nature, 1984. 308(5959): p. 509-513.
12. Pongubala, J.M.R. and M.L. Atchison, PU.1 can participate in an active enhancer complex without its transcriptional activation domain. Proceedings of the National Academy of Sciences, 1997. 94(1): p. 127-132.