Real-time monitoring of a stepwise transcription reaction on a quartz-crystal microbalance

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 (kon) to and the dissociation rate (koff) from the promoter, the proceeding rate (kfor) from the promoter to the forward stall position, the polymerization rate (kcat) of RNA along DNA, and the release rate (kr) from the end of the template DNA. We found that kcat (120 s−1) was extremely large compared with koff (0.014 s−1), kfor (0.062 s−1), and kr (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.