Question

1.WhatarethedifferencesamongRNaseH,RNaseA,RNaseBandRNaseC?2.InyourCDNAkits,RNaseHisaddedinthesecondstrandreactiontoproducemorenickedRNAasprimersforDNAsynthesis.Inthisrepect,wouldRNaseoutRNaseinhibitorinfluencethefunctionofRNaseH,ifitwasaddedevenbeforefirststandsynthesis?

Answer

1.ThemaindifferencebetweenallRNasesarewheretheycleavetheRNA(whatsitetheyrecognize)andwhetheritissinglestrandedordoublestranded.RNaseHisanendoribonucleasethatspecificallyhydorlyzesthephosphodiesterbondsofRNAinRNA:DNAduplexestogenerateproductswith3"hydroxyland5"phosphateends.Itwillnotdegradesingle-strandedordouble-strandedDNAorRNA.RNaseAisanendoribonucleasethatspecificallyhydrolyzesRNAafterCandUresidues.Cleavageoccursbetweenthe3"-phosphategroupofapyrimidineribonucleotideandthe5"-hydroxyloftheadjacentnucleotide.Thereactiongeneratesa2":3"cyclicphosphatewhichthenishydrolyzedtothecorresponding3"nucleosidephosphates.RNaseBisaglycoproteinwhichpossessesanaminoacidcompositionindistinguishablefromthatofRNaseAandwhichcontainscarbohydratetotheextentof6residuesofmannoseand2residuesofN-acetylglucosaminepermolecule.ItisconsequentlyconsideredtobeacarbohydratederivativeofRNaseA.(Tarentino,A.,Plummer,J.,andMaley,F.:StudiesontheOligosaccharideSequenceofRibonucleaseB,J.Biol.Chem.,245,4150(1970).)RNaseBhasthesamespecificityasRNaseA.(Plummer,T.,andHirs,C.:TheIsolationofRibonucleaseB,aGlycoproteinFromBovinePancreaticJuice,J.Biol.Chem.,238,1396(1963).)2.RNASEOUT?inhibitsRNaseA,B,andCbutdoesnotinhibitRNase1,RNaseT1,S1Nuclease,RNaseH,RNaseT2.SoaddingitinfirststrandwillnotcauseaproblemusingRNaseHinsecondstrandsynthesis.