Chen H, Flint S J
Department of Molecular Biology, Princeton University, New Jersey 08544.
J Biol Chem. 1992 Dec 15;267(35):25457-65.
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 nonconserved 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 TA-TAAAA 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.
腺病毒2型IVa2启动子的起始元件足以指导RNA聚合酶II进行准确起始。对体外转录系统中特定碱基对替换对起始活性影响的分析表明，-4至+5位之间的特定序列对起始活性至关重要。损害或消除起始活性的突变改变了序列相关起始子中保守的碱基对和非保守序列。IVa2启动子下游富含TA的序列，以及置于相同下游位点的腺病毒2型主要晚期TATA元件，均无法克服起始子突变的严重抑制作用，这表明起始子是IVa2转录特异性和方向的主要决定因素。相比之下，当ML TATA元件置于IVa2起始子上游31个核苷酸处时，精确的特异性依赖于功能性起始子的存在，但转录效率和方向并非如此。IVa2启动子的活性对富含TA序列的方向或性质变化相对不敏感。此外，只有在IVa2起始子下游包含ML TA-TAAAA序列的启动子才能指导IVa2转录以及来自相反链的转录。本文讨论了这种功能差异对下游元件识别的影响。
