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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Effects of saturation mutagenesis of the phage SP6 promoter on transcription activity, presented by activity logos
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Effects of saturation mutagenesis of the phage SP6 promoter on transcription activity, presented by activity logos

机译：噬菌体SP6启动子的饱和诱变对转录活性的影响，由活性徽标呈现

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摘要

A full set of SP6 promoter variants with all possible single substi- tutions at positions -17 to +5 was constructed. Transcription activities of these variants were individually measured in vivo and in vitro to determine the contribution of each base pair to the promoter activity. The in vivo activity was measured indirectly by transcriptionaI interference of the replication of promoter-bearing pIasmids. This activity depends most highly on residues -11. -9. -8. -7. and + 1 (initiation site). All substitutions at - 11, -9, --8, and -7 abolished formation of closed complexes, except for A -8C. These residues are involved in base-specific interactions with the polymerase, and the substitutions exhibit the same strong inhibi- tion in vitro. In contrast. the in vitro activities of some other variants, measured on linearized templates, were different from those in vivo. Some variants at -13, -4, and -2, among others, showed exceptionally higher activities in vivo than in vitro, sup- porting the possibility that these residues are involved in post- binding steps. including template melting and bending. The A-3T variant showed much lower activity in vivo than in vitro, but it bound to the polymerase 2-fold more than the consensus sequence and is possibly involved in polymerase binding. A quantitative hierarchy of all the base pairs is graphically displayed by activity logos, revealing the energetic contribution of each base pair to the activity.
机译：构建了一整套SP6启动子变体，所有可能的单个取代基位于-17至+5位。在体内和体外分别测量这些变体的转录活性，以确定每个碱基对对启动子活性的贡献。体内活性通过带有启动子的质粒的复制的转录干扰来间接测量。该活性在很大程度上取决于残基-11。 -9。 -8。 -7。和+ 1（初始化站点）。除A -8C外，所有在-11，-9，--8和-7处的取代均消除了封闭络合物的形成。这些残基参与与聚合酶的碱基特异性相互作用，并且这些取代在体外表现出相同的强抑制作用。相反。在线性化模板上测得的其他一些变体的体外活性与体内的不同。在-13，-4和-2处的某些变体在体内的活度要比体外高得多，这支持了这些残基参与结合后步骤的可能性。包括模板熔化和弯曲。 A-3T变体在体内的活性比体外低得多，但它与聚合酶的结合比共有序列高2倍，并且可能参与了聚合酶的结合。通过活动徽标以图形方式显示所有碱基对的定量层次结构，从而揭示每个碱基对对活动的积极贡献。

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