Dual Functioning Genetic Tags for Simultaneous Isolation and Observation of Fluorescent Complexes from Whole Cell Extract

Abstract

Single molecule colocalization methods provide facile means of detecting and analyzing biomolecular complex formation. However, many of these complexes can not be reconstituted from purified systems and must be studied in cell extracts. We have developed a new approach for tagging these complexes with small molecule fluorophores (e.g., Cy3 and Cy5) and for isolation of these complexes in situ during a single molecule experiment. We are able to isolate complexes using either immunoaffinity techniques or by biotin/streptavidin interaction. We have used this method to isolate a number of complexes involved in pre-mRNA splicing in yeast including both the U1 and U6 snRNPs comprising 17 and 8 proteins respectively. By introducing multiple tags, we have analyzed protein stoichiometry in these isolated complexes. Finally, we have performed functional assays on the immobilized snRNPs to study their interactions with other components of the RNA splicing machinery in yeast. We predict that this approach will be broadly applicable for studies of other macromolecular machines.