Transcription factors required for the expression of Xenopus laevis selenocysteine tRNA 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 upstream of the coding region which are functionally interchangeable with the U6 snRNA gene counterparts and an internal B-block, resembling that of classical tRNA genes (1). We have established an in vitro transcription 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) transcription, which could be reconstituted by readdition of partially purified PBP. Addition of increasing amounts of recombinant human TBP to an S100 extract stimulated transcription of the tRNA(Sec), the mouse U6 snRNA and the human Y3 genes, an effect which was not observed in the case of a TATA-less tRNA gene. Purified human TFIIA strongly stimulated tRNA(Sec) transcription in a fashion depending on the concentration of TBP. Surprisingly, partially purified TFIIIC was shown to be dispensable for transcription in vitro and unable to bind the B-block of this gene in vitro, although its sequence matches the consensus for this element. Collectively, these data suggest that the mechanism by which transcription complexes are formed on the tRNA(Sec) gene is dramatically different from that observed for classical tRNA genes and much more resembles that observed for externally controlled pol III genes.

Mammalian transcription factor PBP. Characterization of its binding properties to the proximal sequence element of U6 genes.

The DNA binding properties of human transcription factor PBP, which specifically binds to the proximal sequence element of mammalian U6 genes and which plays a pivotal role during their transcription, were analyzed both qualitatively and quantitatively. As a prerequisite, we analyzed the optimal conditions for DNA binding of the PBP by assaying the stability of the interaction against increasing concentrations of salt, dithiothreitol, and heparin. The protein, which does not induce DNA bending, has a characteristic sensitivity against elevated temperatures and precipitously loses activity between 41 and 43 degrees C, a property which can be used for selective inactivation of the protein. Subjection of the PBP to limited proteinase K treatment showed that the protein consists of at least two functional domains, one of which is required for DNA binding. The PBP binds to the PSE with a much higher specific equilibrium constant (Ks = 1.33 x 10(11) M-1) than to nonspecific DNA (Kn = 1.18 x 10(5) M-1). The association and dissociation rates of PBP.PSE interactions were quantitatively determined by kinetic analyses. The pronounced lag phase during the initiation reaction of mammalian U6 transcription in vitro is probably correlated with the slow binding of the PBP to its target sequence. Once formed, however, the PBP.PSE complex is very stable and has a much lower dissociation (kd = 1.84 x 10(-5) s-1) than association rate constant (ka = 0.18 x 10(6) M-1 s-1). Collectively, the results demonstrate that the PSE binding protein stably associates with a high affinity to its cognate promoter sequence, and this process represents one of the primary events in the formation of the preinitiation complex on the U6 gene. Finally, we analyzed the effect of individual base pair mutations within mammalian U6 PSE sequences on the binding of the PBP.

Identification of transcription factors required for the expression of mammalian U6 genes in vitro.

Transcription factors, required for the basal expression of the mouse U6 gene were identified in extracts from HeLa cells. This gene is transcribed at least four times more efficiently than its human counterpart in extracts from mouse or HeLa cells and hence provides an excellent in vitro system for the identification of transcription factors involved in the basal expression of mammalian U6 genes. At least four separate protein components were found to be required in addition to RNA polymerase III for correct synthesis of U6 RNA in vitro. These correspond to: (i) TFIIIB; (ii) a heat labile activity contained in a protein fraction enriched in TFIID; (iii) an, as yet, uncharacterized component contained in the flow-through upon rechromatography on phosphocellulose, and finally; (iv) a protein specifically binding to the mouse U6 gene promoter and transactivating its expression. Transcription factors IIIA and IIIC are not involved in mammalian U6 transcription in vitro. The U6-specific transcription factor has a molecular mass of approximately 90 +/- 10 kDa. It specifically binds to the U6 gene from bp -42 to -78 on the coding and from bp -37 to -79 on the non-coding strand thereby centrally encompassing the PSE motif of the mouse U6 promoter. The binding activity of this protein is correlated with the efficiency with which the U6 gene is transcribed in vitro, thereby indicating a crucial role of the PSE-binding protein for U6 transcription.