Noguchi M, Miyamoto S, Silverman T A, Safer B
Molecular Hematology Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1994 Nov 18;269(46):29161-7.
We recently discovered an opposing initiator promoter (Inr) downstream of the sense promoter region of the eIF-2 alpha gene (Silverman, T., Noguchi, M., and Safer, B. (1992) J. Biol. Chem. 267, 9738-9742). By reverse transcriptase/polymerase chain reaction analysis of G0 and activated (G1) T-lymphocyte RNAs, overlapping sense and antisense transcripts are now identified. Sense transcription of the eIF-2 alpha gene proceeds from left to right to generate alpha-mRNA; antisense transcription proceeds from right to left to generate RNA, having a sequence complementary to eIF-2 alpha mRNA. Upstream indicates a position 5' relative to the transcription start site. Using DNase I footprint analysis and EMSA, we have found a potential cis-regulatory sequence immediately upstream of the Inr which binds a 43-kDa protein. In addition to conferring protection against DNase I (+457 to +474), the factor also generates hypersensitive sites directly over the Inr (+447 to +457). Insertion of the Inr footprint region into a luciferase reporter gene construct increases expression 150-fold. While mutation of the Inr conserved sequence decreases luciferase activity by 50%, mutation of the 43-kDa factor binding site inhibits luciferase activity by 20%. Sense orientation of the Inr footprint region decreases activity by 80%. The 43-kDa Inr-associated binding protein may be involved in allowing access of RNA polymerase II transcription complexes ot the initiation site of this TATA-less gene. A model for the regulation of eIF-2 alpha expression involving the rapid degradation of dsRNA generated by the relative activities of the two overlapping and opposing promoters is proposed.
我们最近在真核起始因子2α（eIF-2α）基因的有义启动子区域下游发现了一个反向起始子启动子（Inr）（Silverman，T.，Noguchi，M.，和Safer，B.（1992）《生物化学杂志》267，9738 - 9742）。通过对G0期和活化（G1）期T淋巴细胞RNA进行逆转录酶/聚合酶链反应分析，现已鉴定出重叠的有义转录本和反义转录本。eIF-2α基因的有义转录从左向右进行以生成α - mRNA；反义转录从右向左进行以生成与eIF-2α mRNA序列互补的RNA。上游表示相对于转录起始位点的5'位置。使用DNA酶I足迹分析和电泳迁移率变动分析（EMSA），我们在Inr上游紧邻处发现了一个潜在的顺式调控序列，它能结合一种43 kDa的蛋白质。除了对DNA酶I产生保护作用（+457至+474）外，该因子还在Inr正上方（+447至+457）产生超敏位点。将Inr足迹区域插入荧光素酶报告基因构建体中可使表达增加150倍。虽然Inr保守序列的突变使荧光素酶活性降低50%，但43 kDa因子结合位点的突变使荧光素酶活性抑制20%。Inr足迹区域的有义方向使活性降低80%。与Inr相关的43 kDa结合蛋白可能参与使RNA聚合酶II转录复合物能够接近这个无TATA框基因的起始位点。我们提出了一个关于eIF-2α表达调控的模型，该模型涉及由两个重叠且反向的启动子的相对活性产生的双链RNA的快速降解。
