THE RAT VAULT RNA GENE CONTAINS A UNIQUE RNA-POLYMERASE-III PROMOTER COMPOSED OF BOTH EXTERNAL AND INTERNAL ELEMENTS THAT FUNCTION SYNERGISTICALLY

A novel gene transcribed by RNA polymerase (pol) III has been recently identified that produces an RNA component of a large cytoplasmic ribo nucleoprotein complex (Kickhoefer, V. A., Searles, R. P., Kedersha, N. L., Garber, M. E., Johnson, D. L., and Rome, L. H. (1993) J. Biol. Ch em. 268, 7868-7873). Since sequence analysis revealed that this gene c ontains promoter elements from two different classes of RNA pol III ge ne promoters, we examined the function of the 5'-flanking type-3 and i nternal type-2 sequences on transcription activity and the production of stable transcription complexes. We find that the vRNA gene contains a novel RNA pol III promoter, where both the external and internal se quences are essential for template activity and for the productive int eraction of TFIIIC with the internal elements. Thus, the vRNA gene rep resents the first example of a template that requires both type-2 and type-3 promoter elements that appear to function synergistically in th e formation of productive transcription complexes. We have further exa mined the function of the unique arrangement of an internal A box and two B box elements. We find that at least one B element is required fo r template activity. In the absence of the 5'-flanking sequence the pr esence of both B elements inhibits transcription and the binding of TF IIIC. The formation of active complexes is restored when either the B2 element is inactivated or the distance separating the two B elements is increased. Therefore, the B2 element appears to negatively regulate template activity in the absence of the upstream sequences. This uniq ue RNA pol III promoter arrangement may provide a novel mechanism for the regulation of vRNA gene activity.