Purification and characterization of Saccharomyces cerevisiae Rnt1p nuclease.
In this article, we have described methods used to purify Rnt1p and study its biochemical properties. Rnt1p can be easily purified from bacteria as N-terminal His6-tagged protein and its activity may be monitored in vitro. Rnt1p cleaves the RNA by binding to a cleavage site followed by hydrolysis and product release. The kinetic parameters of Rnt1p are similar to those of other nucleases, including bacterial RNase III. The ability of Rnt1p to bind substrate without cleaving it in the absence of divalent metal ions provides a convenient means to study RNA recognition and binding independent of catalysis. The gel mobility shift and in-the-gel cleavage assays described here reveal the formation of two Rnt1p-RNA complexes with different cleavage activities, suggesting that the protein may bind the substrate in two different forms or through a two-step binding reaction.
Lamontagne
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Département de Microbiologie et d'Infectiologie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Elela
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D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D012322:RNA Precursors / Q000737:chemistry; D012335:RNA, Ribosomal / Q000737:chemistry; D043244:Ribonuclease III; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D012322:RNA Precursors; D012335:RNA, Ribosomal; D029701:Saccharomyces cerevisiae Proteins; C065039:RNA, ribosomal, 25S; D006639:Histidine; D004722:Endoribonucleases; C468367:RNT1 protein, S cerevisiae; D043244:Ribonuclease III