Construction and functional analysis of a series of synthetic RNA polymerase III promoters

Abstract

RNA polymerase III promoters were constructed by cloning chemically synthesized double stranded analogues of the box A and box B consensus sequences into suitable vectors. In contrast to approaches adopted previously for the analysis of RNA polymerase III promoters, this method has no limitation on the structure and number of variants generated, and allows critical sequences in various permutations to be studied. Furthermore, the series of synthetic polymerase III promoters created constitute a collection of point mutation variants and hence provide a powerful tool for the analysis of nucleotides essential for promoter function. The results demonstrate that these two boxes, when separated by approximately 50 base pairs, are sufficient to direct efficient transcription, and that substitution of certain nucleotides causes reduced template activity.

The Yeast tRNApheGene Family: Structures and Transcriptional Activities Reveal Member Differences Not Explained by Intragenic Promoters

“…Their role is to provide the physical site for the interaction of transcription factors. For some tRNA genes, the presence of ICRs is sufficient for promoting transcription in vitro, provided they are separated by a central region of 30-70 bp (Ciliberto et al, 1982b;Dingermann et al, 1983;Murphy and Baralle, 1984); in others, the flanking sequences are also important. This is the case for genes from Drosophila for a tRNA2Lys (DeFranco et al, 1980;DeFranco et al 1981), a tRNA^rg (Dingermann et al, 1982), and a tRNAVal (Rajput et al, 1982); genes from Bombyx rnori for a tRNAG|v (Fournier et al, 1984) and a tRNA.Ala (Sprague et al, 1980;Larson et al, 1983;Wilson et al, 1985); a tRNA.M« gene from Xenopus laevis (Hipskind and Clarkson, 1983); and a yeast tRNA3Leu gene (Johnson and Raymond, 1984).…”