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.

“Pre-full-length” transcripts are produced at the end of the polycistronic galactose ( gal ) operon, 5′ galE-galT-galK-galM 3′ , via Rho-dependent transcription termination (RDT) and -independent transcription termination. The 3′ end of the full-length galETKM mRNA is acquired by exonucleolytic processing of the 3′-OH ends of the pre-full-length transcripts. However, the gal operon produces an mRNA termed galE whose 3′ end forms approximately 120 nucleotides downstream of the galE stop codon, within the subsequent gene, galT , thereby establishing polarity in gene expression. In this study, we investigated the molecular processes that generate the 3′ end of galE mRNA. We discovered that the 3′ ends of pre-galE mRNA are produced in the middle of galT as a result of the combination of two separate molecular processes—one previously reported as RDT and the other as unreported RNase E-mediated transcript cleavage. The 3′ ends of pre- galE mRNA undergo exonucleolytic processing to the 3′ end of galE mRNA observed in vivo . A hairpin structure containing an 8 bp stem and a 4-nucleotide loop, located 5–10 nucleotides upstream of the 3′ ends of galE mRNA, blocks exoribonuclease digestion and renders transcript stability. These findings demonstrate that RNase E—contrary to its general role in mRNA degradation—produces RNA 3′ ends that regulate polarity in gene expression. IMPORTANCE: This study reports the findings of two molecular mechanisms that generate the 3′ ends of pre- galE mRNA in the gal operon, viz., Rho-dependent transcription termination and RNase E-mediated cleavage. These 3′ ends are subsequently processed to produce stable galE mRNA with a hairpin structure that prevents exoribonuclease degradation. This mechanism establishes gene expression polarity by generating the 3′ end of galE mRNA within galT in contrast to the usual mRNA degradation role of RNase E. The study reveals a unique role of RNase E in mRNA processing and stability.

In Escherichia coli, transcription is terminated by two mechanisms: Rho-independent termination (RIT) and Rho-dependent termination (RDT). Transcription of the galactose operon is terminated by two tandem terminators, RIT and RDT. In this work, we investigated whether RNA polymerase is terminated randomly at the RIT and RDT sites or is predetermined. Our results show that the transcription and translation complexes have different coupling statuses upon reaching the terminator, of which 50% were loosely coupled and 50% were closely coupled. RIT terminates transcripts that are loosely coupled to translation, and closely coupled transcripts that cannot be terminated by RIT are terminated by the downstream RDT. Thus, RIT and RDT are predetermined events that depend on the coupling status. This rule also holds for the 850 operons in E. coli. Our study reveals an important rule that E. coli has evolved different transcription termination systems at the ends of operons to adapt to different transcription-translation coupling statuses.

The distance between the ribosome and the RNA polymerase active center, often referred to as the mRNA loop length, is a critical determinant of transcription-translation coupling. While structural biology studies have indicated the existence of multiple expressomes with varying mRNA loop lengths, their in vivo roles and functional significance remain largely unexplored. This study delves into the mechanisms governing transcription termination within the Escherichia coli galactose operon, revealing a crucial role in the transcription and translation coupling state. The operon employs both Rho-independent and Rho-dependent terminators. Our findings demonstrate that long-loop coupled transcription-translation complexes preferentially terminate at the upstream Rho-independent terminator. In contrast, short-loop coupled complexes bypass the Rho-independent terminator and terminate at the downstream Rho-dependent terminator. The efficiency of the Rho-independent terminator is enhanced by an extended U-track, suggesting a novel mechanism for overcoming ribosome inhibition. These results challenge the traditional view of transcription termination as a random process, highlighting a predetermined mechanism contingent on the coupling state. This study emphasizes the intricate interactions between transcription and translation in prokaryotes. Understanding how these processes affect the RNA polymerase’s selection of transcriptional terminators is critical for developing strategies to regulate gene expression.

“Pre-full-length” transcripts are produced at the end of the polycistronic galactose ( gal) operon, 5’ galE-galT-galK-galM3’ , via Rho-dependent (RDT) and -independent transcription termination (RIT). The full-length galETKM mRNA’s 3’ end is acquired by exo-nucleolytic processing of the 3’-OH ends of the pre-full-length transcripts. However, the gal operon produces an mRNA named galE whose 3’ end forms at ∼120 nucleotides from the galE stop codon, thus in the following gene, galT , establishing polarity in gene expression. In this study, we investigated the molecular processes that generate the 3’ end of galE mRNA. We discovered that the 3’ ends of pre-galE mRNA are produced in the middle of the galT gene as a result of the combination of two separate molecular processes - one previously reported as RDT and the other as unreported RNase E-mediated transcript cleavage. The 3’ ends of the pre- galE mRNA are exo-nucleolytically processed to the current 3’ end of the galE mRNA. A hairpin structure of 8 base-pair stems and 4 nucleotide-loop formed 5-10 nucleotides upstream of the 3’ ends of the galE mRNA blocks the exoribonuclease digestion and renders stability. These findings showed that RNase E produces RNA 3’end establishing polarity in gene expression, in contrast to the general role of mRNA degradation. Significance statement: Here, we show the findings of two molecular mechanisms that generate the pre- galE mRNA 3’ends in the gal operon: Rho-dependent termination (RDT) and RNase E-mediated cleavage. These 3’ ends are subsequently processed to produce stable galE mRNA with a hairpin structure that prevents exoribonuclease degradation. This mechanism establishes gene expression polarity by generating the 3’ end of galE mRNA within the galT gene, contrasting with the usual mRNA degradation role of RNase E. The study reveals a unique role of RNase E in mRNA processing and stability.