Stereochemistry of polymerization by DNA-dependent RNA-polymerase from Escherichia coli: an investigation with a diastereomeric ATP-analogue.

Abstract

The phosphodiester bond formation by DNA-dependent RNA-polymerase (RNA nucleotidyltransferase, nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) [of E. coli] can in principle result in retention, inversion or racemization of configuration at the .alpha.-P of the nucleoside 5''-triphosphate being polymerized. As a 1st step in elucidating the sterochemistry of this reaction, 1 diastereomer (A) of adenosine 5''-O-(1-thiotriphosphate) (ATP.alpha.S) was polymerized with UTP in the presence of poly(dA-dT.cntdot.poly(dA-dT). The resulting polymer was enzymatically cleaved to uridine 2'',3''-cyclic phosphorothioate which was determined to be the endo-isomer by comparison with an authentic sample. This shows that no racemization occurred and that isomer A of ATP.alpha.S gives a phosphorothioate diester bond with the R-configuration. Whether this represents inversion or retention of configuration awaits elucidation of the absolute configuration of isomer A for ATP.alpha.S.