Stereochemical course of Escherichia coli RNase H.
A new enzymatic method has allowed the assignment of the stereochemistry of E. coli RNase-H-assisted hydrolysis of RNA labelled within the scissile bond with (R(p))-phosphorothioate. This method is based on a stereospecific, two-step enzymatic conversion of cytidine 5'-[(18)O]phosphorothioate into the corresponding 5'-alpha-[(18)O]thiotriphosphate, which is then further used for stereospecific transfer of cytidine 5'-[(18)O]phosphorothioate to the 3'-OH group of a short oligonucleotide with the aid of terminal deoxyribonucleotidyl transferase. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry of the resulting elongated primer revealed that RNase-H-assisted hydrolysis proceeds with inversion of configuration at the phosphorus atom. This result is discussed in the context of current knowledge of the architecture of the active site of the enzyme.
Krakowiak
Agnieszka
A
Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Łódź, Poland.
Owczarek
Alina
A
Koziołkiewicz
Maria
M
Stec
Wojciech J
WJ
D029968:Escherichia coli Proteins; D006868:Hydrolysis; D009841:Oligonucleotides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010104:Oxygen Radioisotopes; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D011849:Radioactive Tracers; D016914:Ribonuclease H / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013237:Stereoisomerism; D013873:Thionucleotides
D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D010104:Oxygen Radioisotopes; D011849:Radioactive Tracers; D013873:Thionucleotides; D012313:RNA; D016914:Ribonuclease H