In vitro transcription of the TATAA-less mouse thymidylate synthase promoter: multiple transcription start points and evidence for bidirectionality

Abstract

The mouse thymidylate synthase (TS) promoter ( pTS) lacks a TATAA box and an initiator element, and has multiple transcription start points ( tsp) located across a 90-bp region. We have developed an in vitro transcription system for pTS using circular templates and nuclear extracts from HeLa cells or mouse 3T6 fibroblasts. The amount of RNA synthesized and the locations of the tsp were determined by S1 nuclease protection assays. The transcription system reproduced the complex pattern of in vivo tsp, except that the downstream tsp were used preferentially. The reaction temperature, concentrations of DNA template and MgCl 2, and incubation time were optimized. The pTS core region contains binding sites for the Sp1 and Ets transcription factors. Inactivation of the Spl-binding element led to a twofold reduction in transcription and a preferential use of upstream tsp. Inactivation of the Ets-binding element, which reduced promoter activity tenfold in vivo, had only a minor effect in vitro. Addition of a strong initiator element introduced a new tsp, but did not eliminate the complex tsp pattern. To determine if pTS had bidirectional promoter activity, the promoter was inverted and analyzed for transcriptional activity. The inverted promoter was found to initiate transcription at multiple tsp and had approximately the same strength as the normal pTS.