Matthew L. Kahlscheuer

Single Molecule Cluster Analysis dissects splicing pathway conformational dynamics

We report Single Molecule Cluster Analysis (SiMCAn), which utilizes hierarchical clustering of hidden Markov modeling–fitted single-molecule fluorescence resonance energy transfer (smFRET) trajectories to dissect the complex conformational dynamics of biomolecular machines. We used this method to study the conformational dynamics of a precursor mRNA during the splicing cycle as carried out by the spliceosome. By clustering common dynamic behaviors derived from selectively blocked splicing reactions, SiMCAn was able to identify the signature conformations and dynamic behaviors of multiple ATP-dependent intermediates. In addition, it identified an open conformation adopted late in splicing by a 3′ splice-site mutant, invoking a mechanism for substrate proofreading. SiMCAn enables rapid interpretation of complex single-molecule behaviors and should prove useful for the comprehensive analysis of a plethora of dynamic cellular machines.

Single-molecule pull-down FRET to dissect the mechanisms of biomolecular machines

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.

Single-Molecule Pull-down FRET (SiMPull-FRET) to dissect the mechanisms of biomolecular machines

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.

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

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.