Reporter Gene-Based qRT-PCR Assay for Rho-Dependent Termination In Vivo.

In bacteria, the Rho protein mediates Rho-dependent termination (RDT) by identifying a non-specific cytosine-rich Rho utilization site on the newly synthesized RNA. As a result of RDT, downstream RNA transcription is reduced. Due to the bias in reverse transcription and PCR amplification, we could not identify the RDT site by directly measuring the amount of mRNA upstream and downstream of RDT sites. To overcome this difficulty, we employed a 77 bp reporter gene , (coding tRNA) from , and we transcriptionally fused it to the sequences to be assayed. We constructed a series of plasmids by combining a segment of the galactose () operon sequences, both with and without the RDT regions at the ends of cistrons (, , and ) upstream of . The RNA polymerase will transcribe the operon sequence and unless it encounters the RDT encoded by the inserted sequence. Since the quantitative real-time PCR (qRT-PCR) method detects the steady state following mRNA synthesis and degradation, we observed that tRNA is degraded at the same rate in these transcriptional fusion plasmids. Therefore, the amount of tRNA can directly reflect the mRNA synthesis. Using this approach, we were able to effectively assay the RDTs and Rho-independent termination (RIT) in the operon by quantifying the relative amount of tRNA using qRT-PCR analyses. The resultant RDT% for , g, and at the end of were 36, 26, and 63, individually. The resultant RIT% at the end of the operon is 33%. Our findings demonstrate that combining tRNA with qRT-PCR can directly measure RIT, RDT, or any other signal that attenuates transcription efficiencies in vivo, making it a useful tool for gene expression research.