The Elongator complex is a unique tRNA acetyltransferase; it was initially annotated as a protein acetyltransferase, but in-depth biochemical analyses revealed its genuine function as a tRNA modifier. The substrate recognition and binding of the Elongator is mainly mediated by its catalytic Elp3 subunit. In this chapter, we describe protocols to generate fluorescently labeled RNAs and outline the principles underlying electrophoretic mobility shift assays (EMSA) and microscale thermophoresis (MST). These two methods allow qualitative and quantitative examinations of the binding affinity of various tRNAs toward the homologs of Elp3 from various organisms. The rather qualitative results from EMSA analyses can be nicely complemented by MST measurements allowing precise determination of the dissociation constant (KD). We also provide detailed notes for users to mitigate potential ambiguities and technical pitfalls during the procedures.
Keywords: Biophysical measurements; EMSA; Elongator; Fluorescent; In vitro transcription; MST; Nonradioactive isotope labeling; tRNA.
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