Abstract
Analyzing proteins as individual molecules by microscopy-based imaging techniques depends on labeling methods. Here, we modified a peptide tag to increase its affinity to sub-nanomolar regime in the context of protein complex and introduced it to RNA polymerase II (pol II) for single-molecule FRET measurement of the proximity between a pol II subunit and TFIIF in the pol II-TFIIF complex. Our localization of a domain in TFIIF through nano-positioning agrees with that based on recent cryo-EM structure by an error less than 10Å. Our work has converted the peptide tag into a versatile tool for extending single-molecule method to protein complexes and illustrates a general approach of designing, incorporating and characterizing a bio-orthogonal moiety in a protein complex.
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