Single-Molecule Pull-Down FRET to Dissect the Mechanisms of Biomolecular Machines.
Single-Molecule Pull-Down FRET to Dissect the Mechanisms of Biomolecular Machines.
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DOI：
10.1016/bs.mie.2015.01.009
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发表时间：
2015
影响因子：
--
通讯作者：
Walter NG
中科院分区：
文献类型：
--
作者：
Kahlscheuer ML;Widom J;Walter NG
Spliceosomes are multi-megadalton RNA-protein complexes responsible for the faithful removal of non-coding 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 towards 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 (TIRF) microscopy in exemplary single molecule pull-down FRET (SiMPull-FRET) studies of the yeast spliceosome and discuss the broad application potential of this technique.
