Fluorescence Reporters for Hfq Oligomerization and RNA Annealing
Fluorescence spectroscopy is a sensitive technique for detecting protein–protein, protein–RNA, and RNA–RNA interactions, requiring only nanomolar concentrations of labeled components. Fluorescence anisotropy provides information about the assembly of multi-subunit proteins, while molecular beacons provide a sensitive and quantitative reporter for base pairing between complementary RNAs. Here we present a detailed protocol for labeling Hfq protein with cyanine 3-maleimide and dansyl chloride to study the protein oligomerization and RNA binding by semi-native polyacrylamide gel electrophoresis (PAGE) and fluorescence anisotropy. We also present a detailed protocol for measuring the rate of annealing between a molecular beacon and a target RNA in the presence of Hfq using a stopped-flow spectrometer.
Key wordsProtein oligomerization RNA chaperone Molecular beacon Fluorescence anisotropy Stopped-flow fluorescence Hfq
The authors thank T. Soper, Y. Peng, and A. Santiago-Frangos for helpful discussion. This work was supported by a grant from the NIH R01 GM46686.