Directed semisynthetic point mutational analysis of an RNA polymerase III promoter

Abstract

The transcription of tRNA and Alu repeat genes in vitro by RNA polymerase III has been shown to be dependent on the presence of two intragenic regions, which contain the consensus sequences RGYNNRRYGG (box A) and GA/TTCRANNC (box B), located 30-60 nucleotides apart. The role of box B and some of its variants was analyzed by a novel method involving the chemical synthesis of double stranded analogues of box B which were subsequently cloned into recombinant vectors carrying box A alone. This method creates a series of semi-synthetic RNA polymerase III promoters and has no limitation on the structure and number of variants which can be generated. The results showed the "wild type" sequence GTTCGAGAC and the sequence GTTCGTGAC (an A to T transversion of the 6th position) were active in promoting RNA polIII transcription. However, the box B sequences CTTCGAGAC and GTACGAGA, where the only departures from the consensus are a G to C and an A to T transversion in the 1st and 3rd positions respectively, were unable to restore promoter function.