Photoregulation of DNA transcription by using photoresponsive T7 promoters and clarification of its mechanism.
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.
Liang
Xingguo
X
Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Japan. liang@mol.nagoya-u.ac.jp
Wakuda
Ryuji
R
Fujioka
Kenta
K
Asanuma
Hiroyuki
H
D001391:Azo Compounds / Q000737:chemistry; D001483:Base Sequence; D019915:DNA Primase / Q000737:chemistry / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005786:Gene Expression Regulation; D007700:Kinetics; D008027:Light; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D055668:Photochemical Processes; D011401:Promoter Regions, Genetic / Q000235:genetics; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000378:metabolism*
D001391:Azo Compounds; D014764:Viral Proteins; D019915:DNA Primase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; C009850:azobenzene