Mastering the control of the Rho transcription factor for biotechnological applications

Villa, Tomás G.; Abril, Ana G.; Sánchez-Pérez, Alicia

doi:10.1007/s00253-021-11326-7

Cited by 2 publications

(1 citation statement)

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“…We speculated that these results are inconsistent for the following reasons: First, most RDTs are regulated by factors other than Rho proteins, including trans-acting elements such as translating ribosomes, RNA-binding proteins, Rho-binding proteins, or cis-acting elements, such as RNA-stem loops, riboswitches, etc. (41)(42)(43). Changes of these factors in the in vivo and in vitro environments can lead to differences in RDT.…”

Section: Possible Reasons For Inconsistency Of Rdt Loci In Vitro and ...mentioning

confidence: 99%

Identification of Rho-dependent termination sitein vivousing synthetic sRNA

Wang

Jeon

et al. 2022

Preprint

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Rho promotes Rho-dependent termination (RDT) at the Rho-dependent terminator, producing a variable-length region at the 3’-end of mRNA without secondary structure. Determining the exact RDT site in vivo is challenging because the 3'-end of mRNA is rapidly removed by 3'- to 5'-exoribonuclease digestion after RDT. Here, we applied synthetic sRNA (sysRNA) to pinpoint RDT sites in vivo by exploiting its complementary base-pairing ability to target mRNA. Through the combined assays of rapid amplification of cDNA 3’-ends, primer extension, and capillary electrophoresis, we could precisely locate 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 seems 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 amounts of mRNA. By constructing a series of sysRNAs with target sites in close proximity, 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 21 bp range, which was determined as RDT site. Our results demonstrated the ability to use sysRNA as a novel tool to precisely localize RDTs in vivo and expanded the range of sysRNA applications.

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Section: Possible Reasons For Inconsistency Of Rdt Loci In Vitro and ...mentioning

confidence: 99%