Ribosome purification approaches for studying interactions of regulatory proteins and RNAs with the ribosome.
Ribosomes are large complexes of RNA and protein that perform the essential task of protein synthesis in the cell. Ribosomes also serve as the initiation point for several translation-associated functions. To perform these tasks efficiently, ribosomes interact with a myriad of nonribosomal proteins and RNAs. Given that most of these interactions are transient, purification of the interacting factors in complex with the ribosome can be a challenging undertaking. Here, we review methods commonly used to isolate ribosomes and study ribosome-associated factors. We also discuss crucial parameters for designing and executing ribosome association studies. Finally, we present a detailed protocol for reporter based enrichment assays that are employed to selectively isolate ribosomes translating a particular message of interest. These protocols can be used to study a wide range of ribosome-associated functions.
Department of Biochemistry and Cell Biology, Stony Brook University, Centers for Molecular Medicine, Stony Brook, NY, USA.
Preeti
P
Mehta
Perry
P
Woo
Krithika
K
Venkataraman
A Wali
AW
Karzai
D002458:Cell Fractionation / Q000379:methods*; D002478:Cells, Cultured; D005095:Exoribonucleases / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D012269:Ribosomal Proteins; D012313:RNA; D005095:Exoribonucleases; C080868:ribonuclease R