Single-Molecule Pull-Down FRET to Dissect the Mechanisms of Biomolecular Machines

Overview

Journal Methods Enzymol

Specialty Biochemistry

Date 2015 Jun 13

PMID 26068753

Citations 3

Authors

Matthew L Kahlscheuer

Julia Widom

Nils G Walter

Affiliations

Soon will be listed here.

Abstract

Spliceosomes are multimegadalton RNA-protein complexes responsible for the faithful removal of noncoding segments (introns) from pre-messenger RNAs (pre-mRNAs), a process critical for the maturation of eukaryotic mRNAs for subsequent translation by the ribosome. Both the spliceosome and ribosome, as well as many other RNA and DNA processing machineries, contain central RNA components that endow biomolecular complexes with precise, sequence-specific nucleic acid recognition, and versatile structural dynamics. Single-molecule fluorescence (or Förster) resonance energy transfer (smFRET) microscopy is a powerful tool for the study of local and global conformational changes of both simple and complex biomolecular systems involving RNA. The integration of biochemical tools such as immunoprecipitation with advanced methods in smFRET microscopy and data analysis has opened up entirely new avenues toward studying the mechanisms of biomolecular machines isolated directly from complex biological specimens, such as cell extracts. Here, we detail the general steps for using prism-based total internal reflection fluorescence microscopy in exemplary single-molecule pull-down FRET studies of the yeast spliceosome and discuss the broad application potential of this technique.

Citing Articles

Coming Together: RNAs and Proteins Assemble under the Single-Molecule Fluorescence Microscope.

Jalihal A, Lund P, Walter N Cold Spring Harb Perspect Biol. 2019; 11(4).

PMID: 30936188 PMC: 6442197. DOI: 10.1101/cshperspect.a032441.

Life under the Microscope: Single-Molecule Fluorescence Highlights the RNA World.

Ray S, Widom J, Walter N Chem Rev. 2018; 118(8):4120-4155.

PMID: 29363314 PMC: 5918467. DOI: 10.1021/acs.chemrev.7b00519.

Single-molecule pull-down for investigating protein-nucleic acid interactions.

Fareh M, Loeff L, Szczepaniak M, Haagsma A, Yeom K, Joo C Methods. 2016; 105:99-108.

PMID: 27017911 PMC: 5408933. DOI: 10.1016/j.ymeth.2016.03.022.

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