Abstract
The genome and transcriptome are constantly modified by proteins in the cell. Recent advances in single-molecule techniques allow for high spatial and temporal observations of these interactions between proteins and nucleic acids. However, due to the difficulty of obtaining functional protein complexes, it remains challenging to study the interactions between macromolecular protein complexes and nucleic acids. Here, we combined single-molecule fluorescence with various protein complex pull-down techniques to determine the function and stoichiometry of ribonucleoprotein complexes. Through the use of three examples of protein complexes from eukaryotic cells (Drosha, Dicer, and TUT4 protein complexes), we provide step-by-step guidance for using novel single-molecule techniques. Our single-molecule methods provide sub-second and nanometer resolution and can be applied to other nucleoprotein complexes that are essential for cellular processes.
Original language | English |
---|---|
Pages (from-to) | 99-108 |
Number of pages | 10 |
Journal | Methods |
Volume | 105 |
DOIs | |
State | Published - 1 Aug 2016 |
Bibliographical note
Funding Information:C.J. was funded by European Research Council under the European Union’s Seventh Framework Programme [FP7/2007-2013]/ERC grant agreement n° [ 309509 ]. We thank V. Narry Kim (Seoul National University), Mikiko Siomi (Keio University), and John Strouboulis (B.S.R.C. Alexander Fleming) for sharing plasmids. We thank Joo lab members for their help.
Funding Information:
C.J. was funded by European Research Council under the European Union's Seventh Framework Programme [FP7/2007-2013]/ERC grant agreement n? [309509]. We thank V. Narry Kim (Seoul National University), Mikiko Siomi (Keio University), and John Strouboulis (B.S.R.C. Alexander Fleming) for sharing plasmids. We thank Joo lab members for their help.
Publisher Copyright:
© 2016 Elsevier Inc.
Keywords
- Dicer
- Drosha
- Protein complex
- RNA interference
- Single-molecule fluorescence
- Single-protein pull-down
- TUT4