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Photoregulation of DNA transcription by using photoresponsive T7 promoters and clarification of its mechanism
Author(s) -
Liang Xingguo,
Wakuda Ryuji,
Fujioka Kenta,
Asanuma Hiroyuki
Publication year - 2010
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2010.07583.x
Subject(s) - azobenzene , t7 rna polymerase , transcription (linguistics) , rna polymerase ii , promoter , dna , chemistry , rna , cis–trans isomerism , rna polymerase , polymerase , microbiology and biotechnology , gene , stereochemistry , biochemistry , biology , gene expression , molecule , organic chemistry , escherichia coli , linguistics , philosophy , bacteriophage
With the use of photoresponsive T7 promoters tethering two 2′‐methylazobenzenes or 2′,6′‐dimethylazobenzenes, highly efficient photoregulation of DNA transcription was obtained. After UV‐A light irradiation (320–400 nm), the rate of transcription with T7 RNA polymerase and a photoresponsive promoter involving two 2′,6′‐dimethylazobenzenes was 10‐fold faster than that after visible light irradiation (400–600 nm). By attaching a nonmodified azobenzene and 2′,6′‐dimethylazobenzene at the two positions, respectively, and by utilizing the different cis → trans thermal stability between cis ‐nonmodified azobenzene and cis‐ 2′,6′‐dimethylazobenzene, four species of T7 promoter ( cis–cis , trans–cis , cis–trans , and trans–trans ) were obtained. The four species showed transcriptional activity in the order of cis–cis > cis–trans > trans–cis > trans–trans . Kinetic analysis revealed that the K m for the cis – cis promoter (both of the introduced azobenzene derivatives were in the cis form) and T7 RNA polymerase was 68 times lower than that for the trans – trans form, indicating that high photoregulatory efficiency was mainly due to a remarkable difference in affinity for RNA polymerase. The present approach is promising for the creation of biological tools for artificially controlling gene expression, and as a photocontrolled system for supplying RNA fuel for RNA‐powered molecular nanomachines.