Nucleic Acids Research

Transcription by T7 RNA polymerase has been studied using a chiral ATP analogue. The Sp diastereoisomer of adenosine 5^0->(1<-thiotriphos" phate) (ATPaS) was incorporated into RNA with an apparent K^ of approximately 15 uM, similar to that for ATP; the Rp diastereoisomer was neither a substrate nor a competitive inhibitor. The configuration of the phosphodiester link in the RNA produced was analyzed with stereospecific nucleases. The rate of nuclease digestion was compared with the rate of digestion of phosphorothioatensubstituted RNA of known stereochemistry synthesized by E.coli RNA polymerase. Surprisingly, the nucleases exhibited reduced discrimination compared with their activity on dinucleotides. The results 3how that phoaphorothioate-nsubstituted RNA transcribed by T7 RNA polymerase has the same configuration as that transcribed by E.coli RNA polymera3e, ie. Rp. Thus, the reaction proceeds with inversion of configuration at phosphorus. INTRODUCTION Chiral analogues of substrate molecules are valuable tools in studying enzyme mechanisms. If the chiral centre is the site of nucleophilic substitution, then the stereochemical course of the reaction can be determined. Reactions involving phosphoryl or nucleotidyl transfer can be analysed using chiral phosphates (1-H). Chiral phosphodiesters and anhydrides can be produced by replacing one of the non^bridging oxygen atoms with either an isotopic oxygen atom or sulphur; in the latter case, a phosphorothioate is formed. These compounds have been used widely to determine the number of elementary steps in enzyme-catalyzed reactions: inversion of configuration results from a single nucleophilic substitution, and retention from a doublcdisplacement reaction, usually indicating the existence of a covalent enzyme intermediate (3,1). Although other reaction courses could, in principle, give rise to either outcome, there is no supporting evidence for these in biological systems. Furthermore, the reaction mechanisms determined for phosphorothioate analogues have been © I R L Press Limited, Oxford, England. 4145 Nucleic Acids Research found to agree with those of phosphates in those cases where analogues containing both typea of chlral linkage have been tested (reviewed in refs. 3 and 1). Chiral nucleoside phosphorothioatea have the further advantage that, whereas one diastereoisomer can be substituted for substrate, the other is frequently found to act as a competitive inhibitor. Thus, stable enzyme-substrate analogue complexes can be formed, or intermediates revealed. For these reasons, deoxynucleoside anthiotriphosphates have been used to determine the atereochemical course of reaction of DNA polymerases (5"9) and internucleotidic phosphorothloates have been used to analyse the mechanisms of reaction of nucleases and a sitenspecific restriction endonucleaae (1O-13). In contrast, very little is known of the mechanisms of sequence-specific reactions involving RNA. In order to use phosphorothioate-substltuted DNA or RNA the stereochemical course of incorporation of dNMPaS or NMPaS must be known. So far all DNA polymerases studied (5-9) and E.coll RNA polymerase (1U) are known to use Sp diastereoisomers of the substrate analogue and proceed with inversion of configuration. However, most RNAs transcribed In vitro are produced U3lng the RNA polymerases of bacterlophages T7 or SP6 (15). Thus, we have sought to determine the stereochemical course of transcription by T7 RNA polymerase, both to see If it follows the general pattern of polymerases and to characterize the RNA produced as a substrate for RNA processing enzymes. In this study, we have used the two diastereoisomers of the substrate ATPaS and nucleasea of known atereospeclficity (previously characterized with respect to small molecules) to characterize both the process of transcription and the stereochemical discrimination of the nucleases. MATERIALS AND METHODS Synthesis of ATPaS and Purification of Diaatereoisomera The aynthesla was performed according to published procedurea (16). Purification by HPLC was performed on a Shlmadzu LC-4A aystem with an Apex 5 u ODS RP column uaing aa eluant 50 mH triethylammonium bicarbonate pH 6.!)5 containing 2J acetonitrile. 31p NMR 3pectroacopy waa performed with broad band proton decoupling on a Bruker AM300 spectrometer operating at 121.5 MHz. Transcription Preparative transcriptions by T7 RNA polymeraae (Boehringer) were