Escherichia coli ribonuclease III: affinity purification of hexahistidine-tagged enzyme and assays for substrate binding and cleavage.
It is now evident that members of the RNase III family of nucleases have central roles in prokaryotic and eukaryotic RNA maturation and decay pathways. Ongoing research is uncovering new roles for RNase III homologs. For example, the phenomena of RNA interference (RNAi) and posttranscriptional gene silencing (PTGS) involve dsRNA processing, carried out by an RNase III homolog. We anticipate an increased focus on the mechanism, regulation, and biological roles of RNase III orthologs. Although the differences in the physicochemical properties of RNase III orthologs, and distinct substrate reactivity epitopes and ionic requirements for optimal activity, may mean that the protocols describe here are not strictly transferrable, the affinity purification methodology, and substrate preparation and use should be generally applicable.
Amarasinghe
A K
AK
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Calin-Jageman
I
I
Harmouch
A
A
Sun
W
W
Nicholson
A W
AW
D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006639:Histidine / Q000737:chemistry*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry; D043244:Ribonuclease III; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D006639:Histidine; D012313:RNA; D004722:Endoribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli