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

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 51 base pairs, are sufficient to direct efficient transcription and that substitution of certain nucleotides causes reduced template activity.