Formation of a template committed complex on the promoter of a gene for the U6 small nuclear RNA from the human requires multiple sequence elements, including the distal region

Abstract

Vertebrate U6 small nuclear RNA (snRNA) loci exemplify a novel class of polymerase III-transcribed genes that lack an intragenic control region (ICR). Instead important transcriptional control elements are located in the 5'-flanking region and resemble those found in promoters and enhancers of polymerase II-transcribed genes. These include a proximal sequence element (PSE), a TATA element, and a distal region containing, at least, an octamer motif. We have used Sarkosyl to characterize steps in U6 promoter transcription in vitro in an unfractionated S100 extract and find very similar properties to those of the adenovirus VA1 gene that contains an ICR. Preformed preinitiation complexes are stable to 0.015% Sarkosyl and can undergo multiple rounds of initiation upon addition of nucleoside triphosphates. A higher concentration (0.075%) prevents reinitiation. In addition, we have investigated the formation of transcription complexes on this promoter in a S100 extract using a template competition assay. No stable complexes are detected with plasmid templates that contain clustered point mutations in the PSE nor with DNAs lacking the U6 5'-flanking region. A plasmid template containing mutations in the TATA element is partially deficient in competitive ability. Furthermore, the distal region upstream of position--148 is necessary for efficient stable complex formation. Within this region, the consensus octamer motif is one component needed to form complexes that withstand competition by a wild-type U6 promoter. The human U2 gene enhancer ligated to the U6 proximal region supports formation of a complex that competes at an intermediate level.