Quantification of Rho-terminationin vivousing qRT-PCR: a comprehensive analysis

Abstract

In prokaryotes, 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 were unable to 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 geneargX, coding transfer RNA that binds L-arginine, tRNAargfromBrevibacterium albidum, and transcriptionally fused it to the sequences to be assayed. We constructed a series of plasmids by combining a segment of the galactose (gal) operon sequences, both with and without the RDT regions at the ends of cistrons (galE,galT, andgalM) upstream ofargX. The RNA polymerase will transcribe thegaloperon sequence andargXunless it encounters the RDT encoded by the inserted sequence. We observed similar tRNAarghalf-lives expressed in these transcriptional fusion plasmids. Therefore, the amount of tRNAargdirectly represents the number of transcripts transcribed. Using this approach, we were able to effectively assay the RDTs in the gal operon by quantifying the relative amount of tRNAargusing quantitative real-time PCR (qRT-PCR) analyses. The resultant RDT% for galET, galTK, and at the end ofgalMwere 36, 26, and 63, individually. Our findings demonstrate that combining tRNAarg, with qRT-PCR can directly measure RDT efficienciesin vivo, making it a useful tool for gene expression research.