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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 Km 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.