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.
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.
It
is well-established that different detection modes
are necessary
for corresponding applications, which can effectively reduce matrix
interference and improve the detection accuracy. Here, we reported
a magnetic separation method based on recombinase polymerase amplification
(RPA)-assisted clustered regularly interspaced short palindromic repeats
(CRISPR)/Cas12a for dual-mode analysis of African swine fever virus
(ASFV) genes, including colorimetry and fluorescence. The ASFV gene
was selected as the initial RPA template to generate the amplicon.
The RPA amplicon was then recognized by CRISPR-associated RNA (crRNA),
activating the trans-cleavage activity of Cas12a
and leading to the nonspecific cleavage of ssDNA as well as a significant
release of alkaline phosphatase (ALP) in the ALP-ssDNA modified magnetic
bead. The released ALP can catalyze para-nitrophenyl
phosphate to generate para-nitrophenol, resulting
in substantial changes in absorbance and fluorescence, both of which
can be used for detection with the naked eye. This strategy allows
the sensitive detection of ASFV DNA, with a 20 copies/mL detection
limit; no cross-reactivity with other viruses was observed. A good
linear relationship was obtained in serum. In addition, this sensor
displayed 100% specificity and sensitivity for clinical sample analysis.
This method integrates the high sensitivity of fluorescence with easy
readout of colorimetry and enables a simple, low-cost, and highly
sensitive dual-mode detection of viral nucleic acid, thereby providing
a broad prospect for the practical application in the diagnosis of
virus infection.
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.
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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