I.U.B:3.1.27.3
RibonucleaseT1isanendoribonuclease,highlyspecificforthecleavageofRNAordeaminatedRNAbetweenguanosine3"-phosphateresidues(orinosine3"-phosphate)andthe5"-OHresiduesofadjacentnucleotideswiththeformationofthecorrespondingintermediate2",3"-cyclicphosphates.Itcleavessingle-strandedRNAreleasingoligonucleotidesfromtheguanosine3"-phosphatetermini.Theenzymehasamolecularweightof11kDa.TheoptimumpHis7.5.RNaseT1isinhibitedbyAg+,Zn2+,Cu2+,andHg2+at1X10-3M.ThestimulatoryeffectsofbothhistidineandEDTAareattributedtochelationofcontaminatinginhibitorcations.TheenzymeassayisessentiallythemethodofEgamietal.,Prog.inNucleicAcidRes.andMolec.Biol.,III,59(1964)baseduponthereleaseofacidsolubleoligonucleotidesfollowingthedigestionofyeastRNA.
Uses:RibonucleaseT1hasextensiveapplicationsinmolecularcloningandDNAsequencing.Becauseofitsspecificityithasbeenacommonlyusedcleavageenzymeforthedeterminationofstructure,nearestneighborfrequencies,andRNAsequencing.Theenzymehasfurtherapplicationinthepreparationofnucleoside2",3"-cyclicphosphates,thesynthesisofoligonucleotides,andtheremovalofRNAfromDNApreparations.Theenzymeisalsousedasanon-mammaliansourceofRNaseinvariousapplications.
StABIlity/Storage:Stable12-18monthsat2-8°C.Storeat2-8°C.
UnitDefinition:OneUnitreleasestheequivalentofoneA260unitofacid-solubleproductsfromyeastRNAin15minutesat37°C,pH7.5
TechnicalNote:Somesuppliersreferencesequencingunits;Onesequencingunitisequivalentto0.075Worthingtonunit.
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