SITES OF RNA POLYMERASE-III TRANSCRIPTION INITIATION AND TY3 INTEGRATION AT THE U6 GENE ARE POSITIONED BY THE TATA BOX

The function of a TATA element in RNA polymerase (EC 2.7.7.6) III tran scription of a naturally TATA-containing U6 snRNA gene and a naturally TATA-less tRNA gene was probed by transcription and Ty3 transposition analyses. Deletion of the TATA box from a U6 minigene did not abolish transcription and Ty3 integration but changed the positions of initia tion and insertion. Insertion of the U6 TATA box at three positions up stream of the TATA-less SUP2 tRNA(Tyr) gene resulted in novel transcri ption initiation and Ty3 integration patterns that depended upon posit ion of the insertion. Nevertheless, the predominant tRNA gene initiati on sites were not affected by insertion of the TATA sequence and remai ned at a fixed distance from the internal box A promoter element. Inse rtions of the TATA box upstream of a SUP2 box A mutant affected the le vel of transcription and restricted the use of upstream start sites, b ut they neither enhanced the use of TATA-dependent initiation sites no r restored expression to the level of the wild-type gene. We conclude that (i) the U6 TATA box is essential in vivo for correct initiation b ut not for transcription, (ii) a TATA box does not compensate for a we ak box A sequence and so cannot perform equivalently, and (iii) the TA TA-binding protein, and probably components of transcription factor II IB, are present on the target at the time of Ty3 integration.