Examination of the rat vRNA gene revealed the presence of sequences characteristic of type-2 and type-3 promoter elements for RNA polymerase III transcribed genes. A typical type-2 gene (exemplified by tRNA genes) contains a split promoter made up of two 10-bp elements, termed the A and B box elements, located within the transcription unit. Interestingly, the rat vRNA contains two B box elements. The rat vRNA gene A box is at position 11- 20 and the two B boxes are at positions 65-75 (B1) and 110-120 (B2), respectively. An RNA polymerase III termination signal consisting of four thymidines is located at the 3' end of the vRNA gene.
Transcription of type-2 genes by RNA polymerase III is dependent on the transcription factors, TFIIIB and TFIIIC, and RNA polymerase III. The A and B boxes serve as binding sites for TFIIIC, which in turn positions TFIIIB immediately upstream of the gene. TFIIIB then directs binding of RNA polymerase III, which initiates transcription. For optimal transcription the A and B box sequences are usually separated by 30-60 bases. The rat vRNA gene is efficiently transcribed by RNA polymerase III in a HeLa S-100 in vitro transcription assay, producing a RNA product of about 140 bases. Unlike most RNA polymerase III transcripts, the rat vRNA has a distinct tissue-specific expression pattern. Basal levels of vRNA are found in all tissues with spleen, intestine, heart, and kidney having the highest levels and brain the lowest level. The mechanism of this tissue-specific expression pattern is unknown. It is possible that the distribution may reveal something about tissue-specific expression of type-2 genes and vault function. Mutational analysis of the vRNA gene promoter suggests that at least one B box is required for transcription of the gene and that the first B box is preferentially used. However, both B boxes are capable of binding to TFIIIC as the mutants defective in either, but not both, are functional in competition assays. Most type-2 genes are not dependent on 5' flanking sequence for efficient transcription. When the 5' flanking sequence is deleted from the vRNA gene, transcription is abolished. This difference may underlie the mechanism of tissue-specific variation. There is a TATA box sequence at postion -25 and a proximal sequence element at position -70 relative to the transcription initiation site. In the absence of the 5' flanking sequence, or at high factor concentrations, the presence of the two B boxes drastically inhibits transcription and stable complex formation, suggesting that the binding of TFIIIC is destabilized in this arrangement. Therefore, it appears that factors interacting in the upstream flanking region may directly participate in enhancing the productive interaction of TFIIIC downstream. This unique arrangement of two closely spaced B boxes in an RNA polymerase III gene may provide a novel mechanism for the regulation of vault RNA gene activity ( Vilalta et al., 1994).
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