Azobenzene-tethered T7 promoter for efficient photoregulation of transcription
Azobenzene-tethered T7 promoter for efficient photoregulation of transcription
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DOI：
10.1021/ja055983k
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发表时间：
2006-01-25
影响因子：
15
通讯作者：
Komiyama, M
中科院分区：
文献类型：
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作者：
Liu, MZ;Asanuma, H;Komiyama, M
在T7启动子的侧链上引入偶氮苯，以光调控T7 RNA聚合酶（T7 RNAP）的转录反应。当在RNAP的环结合区（-7至-11位置）或解旋区（-1至-4位置）将单个偶氮苯分子引入T7启动子时，转录在反式中受到抑制，但在顺式中进行得更快。紫外线照射后的转录物的量相对于在黑暗中的转录物的量是1.5-2.0倍。转录反应的动力学分析表明，在这些位置的光调节机制是不同的。偶氮苯在环结合区的光致异构化主要影响K-m。另一方面，在解旋区域的偶氮苯的异构化主要影响k（cat）。当两个偶氮苯分别被引入到两个环结合和解旋区域时，实现了更明确的光调节：紫外线照射后转录比在黑暗中快7.6倍。只有当两个偶氮苯被引入到这两个不同的区域，分别观察到这种协同效应，并将它们引入到相同的环结合区域大大降低了转录活性。两个偶氮苯在环结合和解旋区域的合作将有助于明确的转录光调控。
Azobenzene was additionally introduced into side chain of T7 promoter for the photocontrol of transcription reaction by T7 RNA polymerase (T7 RNAP). When a single azobenzene molecule was introduced into the T7 promoter either at the loop-binding region of the RNAP (-7 to -11 position) or at the unwinding region (-1 to -4 position), transcription was suppressed in the trans-form but proceeded faster in the cis-form. The amount of transcripts after UV irradiation with respect to that in the dark was 1.5-2.0-fold. Kinetic analysis of the transcription reaction revealed that the photoregulatory mechanism was different in these positions. The photoisomerization of an azobenzene at the loop-binding region primarily affected K-m. On the other hand, the isomerization of an azobenzene at the unwinding region mainly affected k(cat). Still more clear-cut photoregulation was achieved when two azobenzenes were introduced into both loop-binding and unwinding regions, respectively: transcription proceeded 7.6-fold faster after UV irradiation than that in the dark. This synergistic effect was observed only when two azobenzenes were introduced into these two different regions, respectively, and introduction of them into the same loop-binding region drastically lowered the transcription activity. The cooperation of two azobenzenes at loop-binding and unwinding regions would contribute to the clear-cut photoregulation of transcription.