STRUCTURE OF THE TRYPANOSOMA-BRUCEI U6 SNRNA GENE PROMOTER

Transcription in vivo of small nuclear and cytoplasmic RNA genes of Tr ypanosoma brucei was previously shown to require the A and B blocks of a divergently transcribed tRNA or tRNA-like gene located approximatel y 100 nucleotides (nt) upstream. To understand the functioning of thes e transcription units, we have used the U6 snRNA/tRNA(Thr) gents as a model system. Saturation mutagenesis revealed that for transcription i n vivo three elements are essential and sufficient. In addition to the previously described A and B boxes, sequences in the U6 coding region close to the 5' end participate in positioning RNA polymerase III at the start site, and thus constitute a third promoter element. We furth er showed that the function of the upstream A box, but not the B box, is strictly dependent upon its distance to the U6 gene internal contro l region. Using our recently developed transcription extract we furthe r demonstrated that in vitro U6 transcription requires only the intrag enic sequences and the upstream A box of the tRNA(Thr) gene. This appa rent discrepancy between the in vivo and in vitro requirements is high ly reminiscent of U6 snRNA gene transcription in the yeast Saccharomyc es cerevisiae, and suggests the possibility that similar to the yeast system the B block of the trypanosome U6 snRNA gene promoter might be involved in chromatin organization. (C) 1997 Elsevier Science B.V.