A variety of gene expression regulation tools with significant regulatory effects are essential for the construction of complex gene circuits in synthetic biology. Riboswitches have received wide attention due to their unique biochemical, structural, and genetic properties.

In bacteria, small non-coding RNAs (sRNAs) bind to target mRNAs and regulate their translation and/or stability. In the polycistronic galETKM operon of Escherichia coli, binding of the Spot 42 sRNA to the operon transcript leads to the generation of galET mRNA. The mechanism of this regulation has remained unclear. We show that sRNA-mRNA base pairing at the beginning of the galK gene leads to both transcription termination and transcript cleavage within galK, and generates galET mRNAs with two different 3’-OH ends. Transcription termination requires Rho, and transcript cleavage requires the endonuclease RNase E. The sRNA-mRNA base-paired segments required for generating the two galET species are different, indicating different sequence requirements for the two events. The use of two targets in an mRNA, each of which causes a different outcome, appears to be a novel mode of action for a sRNA. Considering the prevalence of potential sRNA targets at cistron junctions, the generation of new mRNA species by the mechanisms reported here might be a widespread mode of bacterial gene regulation.

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 argX, (coding tRNA^arg) from Brevibacterium albidum, and we 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. Since the quantitative real-time PCR (qRT-PCR) method detects the steady state following mRNA synthesis and degradation, we observed that tRNA^arg is degraded at the same rate in these transcriptional fusion plasmids. Therefore, the amount of tRNA^arg can directly reflect the mRNA synthesis. Using this approach, we were able to effectively assay the RDTs and Rho-independent termination (RIT) in the gal operon by quantifying the relative amount of tRNA^arg using qRT-PCR analyses. The resultant RDT% for galET, galTK, and at the end of galM were 36, 26, and 63, individually. The resultant RIT% at the end of the gal operon is 33%. Our findings demonstrate that combining tRNA^arg 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.