Polyadenylic acid addition sites in the adenovirus type 2 major late transcription unit

The cytoplasmic mRNAs which are transcribed from the major late adenovirus promoter can be arranged into five 3'-coterminal families, L1 to L5. We have defined the polyadenylation sites of the mRNAs that belong to the five families at the nucleotide level. From the results, the following conclusions can be made. (i) The hexanucleotide sequence AAUAAA is present at the 3' end of all late adenovirus type 2 mRNAs and precedes the site of polyadenylation by 12 to 30 nucleotides. (ii) Between one and three A residues are present in the genomic sequence at the polyadenylation site. (iii) A sequence with the composition (T)n (A)p (T)q (n, p, q greater than or equal to 1) is found 4 to 24 nucleotides beyond all the adenovirus-specific polyadenylation sites except the 3'-coterminal family L4. This sequence is also found beyond many cellular polyadenylation sites. (iv) The L1 and L2 polyadenylation sites are very similar in structure. The other polyadenylation sites show no apparent sequence relationship, except for the hexanucleotide sequence.     

Transcription from the adenovirus major late promoter uses redundant activating elements

The adenovirus major late promoter (MLP) has been analyzed by constructing recombinant viral genomes containing mutations in possible promoter elements. Single base pair changes in the TATA box had no effect on viral replication, and MLP expression, as measured by the accumulation of late mRNAs, was at wild type levels. However, a double mutation in the TATA box reduced viral replication and MLP expression, demonstrating that the TATA box is important, although not essential, for maximal activity in virus. Primer extension analysis showed that the mRNAs were initiated at the correct position. A mutation in the CAAT box was viable, and had only minor effects on MLP expression. However, this mutation when coupled to a single mutation in the TATA box, severely reduced viral replication and expression from the MLP. Similarly, a viable mutation in the UPE, shown previously to abolish binding of USF, coupled to a single mutation in the TATA box was lethal. These results suggest that both USF and the CAAT box binding factor CP1 can interact with TFIID to effect activation, and thus that the mechanism of activation is functionally redundant.