TRANSCRIPTION FACTORS REQUIRED FOR THE EXPRESSION OF XENOPUS-LAEVIS SELENOCYSTEINE TRANSFER-RNA IN-VITRO

It has previously been reported that transcription in vivo of the tRNA (Sec) gene requires three promoter elements, a PSE and a TATA-box upst ream of the coding region which are functionally interchangeable with the U6 snRNA gene counterparts and an internal B-block, resembling tha t of classical tRNA genes (1). We have established an in vitro transcr iption system from HeLa cells in which three factors, which are either essential for or stimulate transcription were identified. Apart from the TATA-binding protein TBP, the PSE-binding protein PBP was found to be essentially required for expression of the gene. Depletion of PBP from cell extracts by PSE-oligonucleotides abolished tRNA(Sec) transcr iption, which could be reconstituted by readdition of partially purifi ed PBP. Addition of increasing amounts of recombinant human TBP to an S100 extract stimulated transcription of the tRNA(Sec), the mouse U6 s nRNA and the human Y3 genes, an effect which was not observed in the c ase of a TATA-less tRNA gene. Purified human TFIIA strongly stimulated tRNASeC transcription in a fashion depending on the concentration of TBP. Surprisingly, partially purified TFIIIC was shown to be dispensab le for transcription in vitro and unable to bind the B-block of this g ene in vitro, although its sequence matches the consensus for this ele ment. Collectively, these data suggest that the mechanism by which tra nscription complexes are formed on the tRNASeC gene is dramatically di fferent from that observed for classical tRNA genes and much more rese mbles that observed for externally controlled pot III genes.