Cis-acting sequences affecting the length of the poly(A) head of vaccinia virus late transcripts

Mutations in the sequences flanking the conserved and essential TAAAT motif of vaccinia late gene promoters (consensus: T/A T/A TAAAT G Pu Pu) affect the level of expression. Introduction of a pyrimidine in the purine stretch downstream of the TAAAT motif reduces the level of RNA synthesis. Mature transcripts from the wild-type 11K late promoter have a non-contiguous 5′ poly(A) leader of approximately 35 A-residues (referred to as a poly(A) head). We show here by RNA sequencing, primer extension and subsequent m7G cap selection of cDNA/RNA hybrids that the mutations affect the length of the poly(A) head but not the location of the junction between the poly(A) leader and sequences encoded in the genome. These results are consistent with a slippage mechanism underlying the process of 5′ poly(A) addition, but are not in agreement with a splicing event.