Azobenzene-tethered T7 promoter for efficient photoregulation of transcription.
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 Km. On the other hand, the isomerization of an azobenzene at the unwinding region mainly affected kcat. 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.
Liu
Mingzhe
M
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
Asanuma
Hiroyuki
H
Komiyama
Makoto
M
D001391:Azo Compounds / Q000737:chemistry* / Q000378:metabolism; D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D018462:Photobiology; D010777:Photochemistry; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000528:radiation effects*; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D001391:Azo Compounds; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; C009850:azobenzene