Stano Natalie M, Patel Smita S
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
J Biol Chem. 2004 Apr 16;279(16):16136-43. doi: 10.1074/jbc.M400139200. Epub 2004 Feb 5.
Bacteriophage T7 lysozyme binds to T7 RNA polymerase and inhibits transcription initiation and the transition from initiation to elongation. We have investigated each step of transcription initiation to determine where T7 lysozyme has the most effect. Stopped flow and equilibrium DNA binding studies indicate that T7 lysozyme does not inhibit the formation of the preinitiation open complex (open complex in the absence of initiating nucleotide). T7 lysozyme, however, does prevent the formation of a fully open initiation complex (open complex in the presence of the initiating nucleotide). This is consistent with the results that in the presence of T7 lysozyme the rate of G ladder RNA synthesis is about 5-fold slower and the GTP Kd is about 2-fold higher, but T7 lysozyme does not inhibit the initial rate of RNA synthesis with a premelted bulge-6 promoter (bubble from -4 to +2). Neither the RNA synthesis rate nor the extent of promoter opening is restored by increasing the initiating nucleotide concentration, indicating that T7 lysozyme represses transcription by interfering with the formation of a stable and a fully open initiation bubble or by altering the structure of the DNA in the initiation complex. As a consequence of the unstable initiation bubble and/or the inhibition of the conformational changes in the N-terminal domain of T7 RNAP, T7 lysozyme causes an increased production of abortive products from 2- to 5-mer that delays the transition from the initiation to the elongation phase.
噬菌体T7溶菌酶与T7 RNA聚合酶结合，抑制转录起始以及从起始到延伸的转变。我们研究了转录起始的每一步，以确定T7溶菌酶在哪一步影响最大。停流和平衡DNA结合研究表明，T7溶菌酶不抑制起始前开放复合物（不存在起始核苷酸时的开放复合物）的形成。然而，T7溶菌酶确实会阻止完全开放的起始复合物（存在起始核苷酸时的开放复合物）的形成。这与以下结果一致：在存在T7溶菌酶的情况下，G梯状RNA合成速率慢约5倍，GTP的解离常数高约2倍，但T7溶菌酶不抑制使用预熔解的凸起-6启动子（从-4到+2的气泡）时RNA合成的初始速率。增加起始核苷酸浓度既不能恢复RNA合成速率，也不能恢复启动子开放程度，这表明T7溶菌酶通过干扰稳定且完全开放的起始气泡的形成或改变起始复合物中DNA的结构来抑制转录。由于起始气泡不稳定和/或T7 RNA聚合酶N端结构域构象变化受到抑制，T7溶菌酶导致2至5聚体流产产物的产量增加，从而延迟了从起始阶段到延伸阶段的转变。
