Messenger RNA for the Ad2 DNA binding protein: DNA sequences encoding the first leader and heterogenity at the mRNA 5' end

Abstract

During the early stage of Ad2 infection of human cells, RNA is transcribed from five separate transcription units. Early region II encodes the mRNA for a 72K single-stranded DNA binding protein (DBP) which functions in DNA replication. This report describes the structure of the first leader of the DBP mRNA and the flanking sequences in the DNA. The leader, labeled in vivo with 32P, was isolated by DNA filter hybridization to the viral restriction fragment Eco RI F, and its RNAase T1 and RNAase A oligonucleotides were analyzed by RNA fingerprinting techniques. Comparison of this RNA sequence information with the DNA sequence of Eco RI F has located a 68 nucleotide region of the Hae III C subfragment at coordinate 75.1 that encodes the leader. This position is near the coordinate to which nascent chain analysis and ultraviolet transcription mapping have mapped an RNA initiation site, or promoter, for the DBP mRNA. The DNA sequence that overlaps the leader on the 3' side contains a donor sequence for splicing this leader to a second downstream leader. The splicing sequence shows a seven base homology with the comparable structure of the Ad2 major late leader, and a mouse globin mRNA splicing sequence. The DNA sequence upstream from the cap, the region oof the potential promoter site does not, however, contain a "TA-TAAA"-type homology of the sort noted by D. Hogness, M. Goldberg and R. Lifton (personal communication) for many cellular transcription units, and by other investigations for the Ad2 major late transcription unit. Also, the leader is found with two distinct capped 5' termini, 7meGpppA and 7meGpppG, which are encoded at adjacent positions in the DNA and thus are from mRNAs which are staggered by one nucleotide in length at the 5' end. The staggering at the 5' terminus and the lack of the upstream homolgy distinguish the DBP mRNA from many viral and cellular messenger. In both these respects, however, the DBP mRNA resembles the late messengers of SV40 and polyoma viruses. In this paper, we discuss the implications of these findings for the mechanism of specifying mRNA 5' ends.