title: Real-time monitoring of a stepwise transcription reaction on a quartz-crystal microbalance.
abstract: We monitored real-time DNA transcription by T7 RNAP using a 27-MHz DNA-immobilized quartz-crystal microbalance (QCM) in buffer solution to investigate the stepwise reaction of transcription. We designed a template double-stranded DNA that consisted of a T7 promoter, a stall position (15 bp downstream from the promoter), and a 73-bp transcription region. Based on the frequency (mass) changes of the template-immobilized QCM in response to the addition of T7 RNAP and monomers of NTP, we obtained the kinetic parameters of each step of the T7 RNAP reactions: the enzyme-binding rate (k(on)) to and the dissociation rate (k(off)) from the promoter, the proceeding rate (k(for)) from the promoter to the forward stall position, the polymerization rate (k(cat)) of RNA along DNA, and the release rate (k(r)) from the end of the template DNA. We found that k(cat) (120 s⁻¹) was extremely large compared with k(off) (0.014 s⁻¹), k(for) (0.062 s⁻¹), and k(r) (0.014 s⁻¹), revealing that the rate-limiting steps of T7 RNAP involve the binding to the promoter, the movement to the stall position, and the release from DNA. These kinetic parameters were compared with values for other DNA-binding enzymes.
authors.affiliation: Department of Biomolecular Engineering and Global Centre of Excellence, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
authors.forename: Shuntaro
authors.initials: S
authors.lastname: Takahashi
authors.forename: Kazuya
authors.initials: K
authors.lastname: Hisanaga
authors.forename: Aya
authors.initials: A
authors.lastname: Yoshida
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Okahata
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D007700:Kinetics; D011401:Promoter Regions, Genetic; D058525:Quartz Crystal Microbalance Techniques; D013997:Time Factors; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry
chemical_list: D017931:DNA Primers; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases