Identification of a Rho-Dependent Termination Site
            <i>In Vivo</i>
            Using Synthetic Small RNA

Wang, Xun; N, Monford Paul Abishek; Jeon, Heung Jin; He, Jin; Lim, Heon M.

doi:10.1128/spectrum.03950-22

Cited by 4 publications

(8 citation statements)

References 61 publications

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“…Still, researchers should be aware of its limitations and select the best method suited to their experimental design and research question. Traditional methods for studying RDT, such as Northern blotting and primer extension assays, may be less sensitive, quantitative, and time-consuming (1,6,8,10,21,22,33). These methods may also necessitate more RNA and are unsuitable for high-throughput analysis.…”

Section: Discussionmentioning

confidence: 99%

“…The copyright holder for this preprint (which this version posted April 11, 2023. ; https://doi.org/10.1101/2023.04.11.536429 doi: bioRxiv preprint through genetic and biochemical experiments, several issues make predicting and determining RDT regions in vivo still difficult (3,6,7), including (i) mechanism complexity of RDT involves multiple components, including RNAP, the nascent RNA transcript, and the Rho protein. The interactions between these components make understanding the mechanism of RDT difficult; (ii) Rho is not specific to any mRNA sequence and can bind to any RNA sequence containing a Rho utilization (rut) site; (iii) the instability of Rho-terminated RNA 3' ends: the 3' end of Rho-terminated mRNA is rapidly digested by 3' to 5' exonuclease digestion after RDT due to the lack of secondary structure.…”

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confidence: 99%

“…Our previous research recognized three RDTs that span the galE-galT and galT-galK cistron junctions, the first and second structural genes of the galactose operon, as well as downstream of galM, the last structural gene of the gal operon (1,(8)(9)(10). Using synthetic small RNAs (sysRNAs), we were also able to identify the in vivo RDT site downstream of galM (6), however, we were unable to identify the RDT sites covering the galE-galT and galT-galK cistron junctions, possibly due to ribosome and sysRNA competition. Therefore, to gain a deeper insight into the RDT mechanism, it is necessary to carefully consider these issues and apply more widely applicable techniques for RDT identification.…”

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confidence: 99%

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

Jeon

Wang³

et al. 2023

Preprint

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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.

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Section: Discussionmentioning

confidence: 99%

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

Jeon

Wang³

et al. 2023

Preprint

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“…Several issues make studying RDT difficult: Rho is not specific to any mRNA sequence and can bind to any RNA sequence containing a Rho utilization (rut) site, and the formation of stable RNA secondary structures can interfere with Rho binding to the rut site, resulting in inefficient or incomplete transcription termination. Using synthetic small RNAs (sysRNAs), we were also able to identify the in vivo RDT site downstream of galM [7]; however, we were unable to identify the RDT sites covering the galE-galT and galT-galK cistron junctions, possibly due to ribosome and sysRNA competition. Therefore, to gain a deeper insight into the RDT mechanism, it is necessary to carefully consider these issues and apply more widely applicable techniques for RDT identification.…”

Section: Introductionmentioning

confidence: 86%

“…In Escherichia coli, Rho is essential for survival, and about >20-30% of its genes are terminated by Rho, indicating its importance in Escherichia coli [4,5]. Although researchers have been studying the working mechanisms of Rho for decades through genetic and biochemical experiments, several issues make predicting and determining RDT regions in vivo still difficult [3,6,7], including the fact that (i) the mechanism complexity of RDT involves multiple components, including RNAP, the nascent RNA transcript, and the Rho protein. The interactions between these components make understanding the mechanism of RDT difficult; (ii) Rho is not specific to any mRNA sequence and can bind to any RNA sequence containing a rut site; (iii) the instability of Rho-terminated RNA 3 ends: the 3 end of Rho-terminated mRNA is rapidly digested by 3 to 5 exonuclease digestion after RDT due to the lack of a secondary structure.…”

Section: Introductionmentioning

confidence: 99%

Reporter Gene-Based qRT-PCR Assay for Rho-Dependent Termination In Vivo

Jeon,

Wang

et al. 2023

Cells

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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.

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