Quantification of Rho-termination in vivo using 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 gene argX, coding transfer RNA that binds L-arginine, tRNAarg from Brevibacterium 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, and galM) upstream of argX. The RNA polymerase will transcribe the gal operon sequence and argX unless it encounters the RDT encoded by the inserted sequence. We observed similar tRNAarg half-lives expressed in these transcriptional fusion plasmids. Therefore, the amount of tRNAarg directly 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 tRNAarg using quantitative real-time PCR (qRT-PCR) analyses. The resultant RDT% for galET, galTK, and at the end of galM were 36, 26, and 63, individually. Our findings demonstrate that combining tRNAarg, with qRT-PCR can directly measure RDT efficiencies in vivo, making it a useful tool for gene expression research.