Point mutations in the regulatory region of the human adenoviral VAI gene

Abstract

The VA RNA genes of human adenoviruses are transcribed by RNA polymerase III and, like other RNA polymerase III transcriptional units, contain internal regulatory regions. By means of a scheme for directed mutagenesis which employs a deletion mutation to define the target, point mutations have been induced in the regulatory region of a cloned VAI gene. One mutant plasmid which fails to direct VAI transcription and several others which direct the synthesis of normal amounts of RNA of aberrant electrophoretic mobilities were identified using an in vitro transcription assay. The nucleotide sequences of the regulatory regions of three of these plasmids have been determined. All three contain mutations which fall in a block of nucleotides whose sequence (GGTTCGANNCC) is highly conserved in RNA polymerase III promoters (Galli, G., Hofstetter, H., and Birnstiel, M. L. (1981) Nature (Lond.) 294, 626-631). The VAI-negative mutant (pRR5028) contains a G leads to A transition mutation at the second position, which is invariant in functional RNA polymerase III promoters. One mobility variant carries three point mutations, including one G leads to A base change at the position adjacent on the 5' side to that altered in pRR5028. The third mutant contains a single C leads to T transition mutation, in the final position of the conserved block.