The coding gene of T7 RNAP was amplified from the genomic DNA of E.
Subsequently, T7 RNAP gene was cloned into the BamH I and Xho I restriction sites of lentiviral vector pLEX-MCS and prokaryotic expression vector pET-32a (+) (Novagen, USA) to yield the pLEX-T7 RNAP and pET-32a-T7 RNAP recombinant plasmids.
Lentivirus-mediated T7 RNAP transcationpLEX-MCS, pLEX-T7 RNAP and pLEX- EGFP plasmids were prepared using a Endofree Maxi Plasmid Kit (Tiangen, China) and co- transfected with the helper plasmids (pSPAX2 and pMD2.
Prokaryotic expression of T7 RNAPTo identify the protein molecular weight ofT7 RNAP, T7 RNAP gene was cloned into a prokaryotic expression vector pET-32a (+) and SDS-PAGE was performed to examine the protein of T7 RNAP.
Lentiviral-mediated T7 RNAP intergrationLentivirus was introduced to integrate T7 RNAP into MDBK cells.
3A, the 2-Ct value of T7 RNAP and EGFP expression levels were expected to be close to 1, suggesting that the positive rate of MDBK-T7 and MDBK-EGFP cells were almost near to 100%.
So, molecular motors such as RNAP could work like microscopic Maxwell demons, using energy to select favorable fluctuations of energy when opportunities arise.
When they decoded RNAP's structure, Kornberg and his team discovered that RNAP includes a system of two moving parts, located next to the site within RNAP where new RNA bases bind to the DNA template.
Indeed, in 2005, Nudler and his collaborators showed that mutations altering the trigger structure rendered the RNAP unable to move preferentially forward.
Block's team measured the pull exerted by single RNAP molecules during the transcription process.
So, Brownian motion would provide the energy for RNAP to crawl along DNA.
GEOGRAPHY AS DESTINY No matter what the details of its machinery are, RNAP is an example of how evolution has invented ways of doing complex tasks in the forbidding environment of Brownian motion.