UPSTREAM PROMOTER ELEMENTS ARE SUFFICIENT FOR SELENOCYSTEINE TRNA([SER]SEC) GENE-TRANSCRIPTION AND TO DETERMINE THE TRANSCRIPTION START POINT

The TATA box, located upstream at about nt -30, and the proximal seque nce element, located at about nt -60, are both essential and sufficien t for basal level transcription of the Xenopus laevis (Xl) selenocyste ine (Sec) tRNA([Ser]Sec) gene as demonstrated by its microinjection in to Xl oocytes. Point mutations within either of these regions abolish transcription, while deletion of the internal boxA dement or insertion of 13 nt within the internal boxB element does not impair transcripti on, The latter mutations (within the internal regions) affect processi ng of the 3'-trailer sequence. Replacement of the tRNA([Ser]Sec) codin g sequence with an Escherichia coli M1 RNA gene resulted in expression of the E. coli gene governed by the upstream tRNA([Ser]Sec) promoter elements. These studies demonstrate unequivocally that the upstream pr omoter elements are sufficient for the basal level of tRNA([Ser]Sec) g ene transcription. Primer extension studies with spacer mutants show t hat the internal elements do not play a role in selecting the transcri ption start point (tsp), but that selection of the tsp is determined b y the region upstream from the gene. Further, studies with spacer muta nts show that the distance between the TATA box and the tsp is quite l ikely the critical factor in selecting the position of tsp.