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 argX, (coding tRNAarg) 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 tRNAarg is degraded at the same rate in these transcriptional fusion plasmids. Therefore, the amount of tRNAarg 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 tRNAarg 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 tRNAarg 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.

sRNA expedites polycistronic mRNA decay in Escherichia coli.

In bacteria, most small RNA (sRNA) elicits RNase E-mediated target mRNA degradation by binding near the translation initiation site at the 5' end of the target mRNA. Spot 42 is an sRNA that binds in the middle of the gal operon near the translation initiation site of galK, the third gene of four, but it is not clear whether this binding causes degradation of gal mRNA. In this study, we measured the decay rate of gal mRNA using Northern blot and found that Spot 42 binding caused degradation of only a specific group of gal mRNA that shares their 3' end with full-length mRNA. The results showed that in the MG1655Δspf strain in which the Spot 42 gene was removed, the half-life of each gal mRNA in the group increased by about 200% compared to the wild type. Since these mRNA species are intermediate mRNA molecules created by the decay process of the full-length gal mRNA, these results suggest that sRNA accelerates the mRNA decaying processes that normally operate, thus revealing an unprecedented role of sRNA in mRNA biology.

Identification of a Rho-Dependent Termination Site In Vivo Using Synthetic Small RNA.

Rho promotes Rho-dependent termination (RDT) at the Rho-dependent terminator, producing a variable-length region without secondary structure at the 3' end of mRNA. Determining the exact RDT site in vivo is challenging, because the 3' end of mRNA is rapidly removed after RDT by 3'-to-5' exonuclease processing. Here, we applied synthetic small RNA (sysRNA) to identify the RDT region in vivo by exploiting its complementary base-pairing ability to target mRNA. Through the combined analyses of rapid amplification of cDNA 3' ends, primer extension, and capillary electrophoresis, we could precisely map and quantify mRNA 3' ends. We found that complementary double-stranded RNA (dsRNA) formed between sysRNA and mRNA was efficiently cleaved by RNase III in the middle of the dsRNA region. The formation of dsRNA appeared to protect the cleaved RNA 3' ends from rapid degradation by 3'-to-5' exonuclease, thereby stabilizing the mRNA 3' end. We further verified that the signal intensity at the 3' end was positively correlated with the amount of mRNA. By constructing a series of sysRNAs with close target sites and comparing the difference in signal intensity at the 3' end of wild-type and Rho-impaired strains, we finally identified a region of increased mRNA expression within the 21-bp range, which was determined as the RDT region. Our results demonstrated the ability to use sysRNA as a novel tool to identify RDT regions in vivo and expand the range of applications of sysRNA. IMPORTANCE sysRNA, which was formerly widely employed, has steadily lost popularity as more novel techniques for suppressing gene expression come into existence because of issues such as unstable inhibition effect and low inhibition efficiency. However, it remains an interesting topic as a regulatory tool due to its ease of design and low metabolic burden on cells. Here, for the first time, we discovered a new method to identify RDT regions in vivo using sysRNA. This new feature is important because since the discovery of the Rho protein in 1969, specific identification of RDT sites in vivo has been difficult due to the rapid processing of RNA 3' ends by exonucleases, and sysRNA might provide a new approach to address this challenge.

Failure of Translation Initiation of the Next Gene Decouples Transcription at Intercistronic Sites and the Resultant mRNA Generation.

In Escherichia coli, transcription is coupled with translation. The polar gal operon is transcribed galE-galT-galK-galM; however, about 10% of transcription terminates at the end of galE because of Rho-dependent termination (RDT). When galE translation is complete, galT translation should begin immediately. It is unclear whether RDT at the end of galE is due to decoupling of translation termination and transcription at the cistron junction. In this study, we show that RDT at the galE/galT cistron junction is linked to the failure of translation initiation at the start of galT, rather than translation termination at the end of galE. We also show that transcription pauses 130 nucleotides downstream from the site of galE translation termination, and this pause is required for RDT. IMPORTANCE Transcription of operons is initiated at the promoter of the first gene in the operon, continues through cistron junctions, and terminates at the end of the operon, generating a full-length mRNA. Here, we show that Rho-dependent termination of transcription occurs stochastically at a cistron junction, generating a stable mRNA that is shorter than the full-length mRNA. We further show that stochastic failure in translation initiation of the next gene, rather than the failure of translation termination of the preceding gene, causes the Rho-dependent termination. Thus, stochastic failure in translation initiation at the cistron junction causes the promoter-proximal gene to be transcribed more than promoter-distal genes within the operon.

An in vitro Assay of mRNA 3' end Using the E. coli Cell-free Expression System.

At the end of about 80% of the operon in Escherichia coli, translation termination decouples transcription, leading to Rho-dependent transcription termination (RDT). However, no in vitro or in vivo assay system has proven to be good enough to see the 3' end of the mRNA generated by RDT. Here, we present a cell-free assay system that could provide detailed information on the 3' end of a transcript RNA generated by RDT. Our protocol shows how to extract transcript RNA generated by transcription reactions from a cell-free extract, followed by an RNA oligomer ligation to the 3' end of a transcript RNA of interest. The 3' end of the RNA is amplified using RT-PCR. Its genetic location can be determined using a gene-specific primer extension reaction. The 3' ends of mRNA can be visualized and quantified by polyacrylamide gel electrophoresis. One significant advantage of a cell-free assay system is that factors involved in the generation of the 3' end, such as proteins and sRNA, can be directly assayed by exogenously adding factor(s) to the reaction. Graphic abstract: An illustration of the experimental methodology.

sRNA-mediated regulation of gal mRNA in E. coli: Involvement of transcript cleavage by RNase E together with Rho-dependent transcription termination.

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.

Translation Initiation Control of RNase E-Mediated Decay of Polycistronic gal mRNA.

In bacteria, mRNA decay is a major mechanism for regulating gene expression. In Escherichia coli, mRNA decay initiates with endonucleolytic cleavage by RNase E. Translating ribosomes impede RNase E cleavage, thus providing stability to mRNA. In transcripts containing multiple cistrons, the translation of each cistron initiates separately. The effect of internal translation initiations on the decay of polycistronic transcripts remains unknown, which we have investigated here using the four-cistron galETKM transcript. We find that RNase E cleaves a few nucleotides (14-36) upstream of the translation initiation site of each cistron, generating decay intermediates galTKM, galKM, and galM mRNA with fewer but full cistrons. Blocking translation initiation reduced stability, particularly of the mutated cistrons and when they were the 5'-most cistrons. This indicates that, together with translation failure, the location of the cistron is important for its elimination. The instability of the 5'-most cistron did not propagate to the downstream cistrons, possibly due to translation initiation there. Cistron elimination from the 5' end was not always sequential, indicating that RNase E can also directly access a ribosome-free internal cistron. The finding in gal operon of mRNA decay by cistron elimination appears common in E. coli and Salmonella.

Processing generates 3' ends of RNA masking transcription termination events in prokaryotes.

Two kinds of signal-dependent transcription termination and RNA release mechanisms have been established in prokaryotes in vitro by: (i) binding of Rho to cytidine-rich nascent RNA [Rho-dependent termination (RDT)], and (ii) the formation of a hairpin structure in the nascent RNA, ending predominantly with uridine residues [Rho-independent termination (RIT)]. As shown here, the two signals act independently of each other and can be regulated (suppressed) by translation-transcription coupling in vivo. When not suppressed, both RIT- and RDT-mediated transcription termination do occur, but ribonucleolytic processing generates defined new 3' ends in the terminated RNA molecules. The actual termination events at the end of transcription units are masked by generation of new processed 3' RNA ends; thus the in vivo 3' ends do not define termination sites. We predict generation of 3' ends of mRNA by processing is a common phenomenon in prokaryotes as is the case in eukaryotes.

Visualization of RNA 3' ends in Escherichia coli Using 3' RACE Combined with Primer Extension.

In this assay, 3' RACE (Rapid Amplification of cDNA 3' Ends) followed by PE (primer extension), abbreviated as 3' RACE-PE is used to identify the mRNA 3' ends. The following protocol describes the amplification of the mRNA 3' ends at the galactose operon in E. coli and the corresponding visualization of the PCR products through PE. In PE, the definite primer is 5' end-labeled using [γ-(32) P] ATP and T4 polynucleotide kinase, which anneals to the specific DNA molecules within the PCR product of the 3' RACE. The conventional PE can only be used to locate the 5' end of an mRNA transcript since reverse transcriptase (RTase) polymerizes only in the 5' → 3' direction. Thus, Taq polymerase is used instead of RTase, PCR is performed. Therefore, we are able to locate the 3' end of the mRNA using this assay. The relative amount of the 3' end can be directly visualized and quantified by way of separating DNA products in a denaturing 8% urea-PAGE (Polyacrylamide Gel Electrophoresis) gel. The exact position of the 3' ends can be sequenced by comparison of these final DNA products with the corresponding DNA sequencing ladder.