Orientation-Dependent Transcriptional Activator Upstream of a Human U2 snRNA Gene

Abstract

We examined the structure of the promoter for the human U2 snRNA gene, a strong RNA polymerase II transcription unit without an obvious TATA box. A set of 5′ deletions was constructed and assayed for the ability to direct initiation of U2 snRNA after microinjection into Xenopus oocytes. Sequences between positions −295 and −218 contain an activator element which stimulates accurate initiation by 20- to 50-fold, although as few as 62 base pairs of 5′ flanking sequence are sufficient to direct the accurate initiation of U2 RNA. When the activator was recloned in the proper orientation at either of two different upstream locations, the use of the normal U2 start site was stimulated. Inversion of the element destroyed the stimulation of accurate U2 initiation, but initiation at aberrant upstream start sites was enhanced by the element in both orientations. A 4-base-pair deletion that destroyed the activity of the element lies within a sequence (region III) which is highly conserved among U2 genes from different organisms. Mutations in the activator also affected the ability of the U2 template to compete with a wild-type U1 gene in coinjection experiments. We propose that the element enhances the efficiency of transcription in part by facilitating the association of a limiting factor with transcription complexes. Human U1 snRNA genes possess a region homologous to U2 region III, and we suggest that upstream activator elements may be a general feature of snRNA promoters.