Mutational analysis of the adenovirus 2 IVa₂ initiator and downstream elements

The initiator element of the adenovirus type 2 IVa₂ promoter is sufficient to direct accurate initiation by RNA polymerase II. Analysis of the effects of substitution of specific base pairs on initiator activity in in vitro transcription systems indicated that specific sequences between positions -4 and +5 were essential for initiator activity. Mutations that impaired or eliminated initiator activity altered both base pairs that are conserved in sequence-related initiators and non-conserved sequences. Neither the downstream TA-rich sequence of the IVa₂ promoter, nor the adenovirus 2 major late TATA element placed at the same downstream site could overcome the severe inhibitory effects of initiator mutations, indicating that the initiator is the primary determinant of the specificity and direction of IVa₂ transcription. By contrast, when the ML TATA element was placed 31 nucleotides upstream of the IVa₂ initiator, the precise specificity, but neither the efficiency nor direction of transcription, depended on the presence of a functional initiator. Activity of the IVa₂ promoter was relatively insensitive to changes in the orientation or nature of the TA-rich sequence. Furthermore, only a promoter containing the ML TATAAAA sequence downstream of the IVa₂ initiator was competent to direct both IVa₂ transcription and transcription from the opposite strand. The implications of this functional difference for recognition of the downstream element are discussed.