Shine-Dalgarno sequence enhances the efficiency of lacZ repression by artificial anti-lac antisense RNAs in Escherichia coli

Journal of Bioscience and Bioengineering
Alessandra StefanAlejandro Hochkoeppler

Abstract

Silencing of the lacZ gene in Escherichia coli was attempted by means of the expression of antisense RNAs (asRNAs) in vivo. A short fragment of lacZ was cloned into the pBAD expression vector, in reverse orientation, using the EcoRI and PstI restriction sites. This construct (pBAD-Zcal1) was used to transform E. coli cells, and the antisense transcription was induced simply by adding arabinose to the culture medium. We demonstrated that the Zcal1 asRNA effectively silenced lacZ using β-galactosidase activity determinations, SDS-PAGE, and Western blotting. Because the concentration of the lac mRNA was always high in cells that expressed Zcal1, we hypothesize that this antisense acts by inhibiting messenger translation. Similar analyses, performed with a series of site-specific Zcal1 mutants, showed that the Shine-Dalgarno sequence, which is conferred by the pBAD vector, is an essential requisite for silencing competence. Indeed, the presence of the intact Shine-Dalgarno sequence positively affects asRNA stability and, hence, silencing effectiveness. Our observations will contribute to the understanding of the main determinants of silencing as exerted by asRNAs as well as provide useful support for the design of robust and effici...Continue Reading

References

Apr 1, 1974·Proceedings of the National Academy of Sciences of the United States of America·J Shine, L Dalgarno
Jun 1, 1974·Proceedings of the National Academy of Sciences of the United States of America·W S ReznikoffC K Hurley
Dec 1, 1984·Proceedings of the National Academy of Sciences of the United States of America·S PestkaR K Pestka
Jun 30, 1995·Journal of Molecular Biology·A P GultyaevC W Pleij
Feb 12, 1998·Nucleic Acids Research·P De Rijk, R De Wachter
May 26, 2005·Trends in Genetics : TIG·Susan Gottesman
Oct 27, 2005·Current Topics in Medicinal Chemistry·Zhanna V ZhilinaScot W Ebbinghaus
Aug 30, 2008·BMB Reports·Stefan AlessandraHochkoeppler Alejandro

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Citations

Feb 21, 2014·International Journal of Molecular Sciences·Nobutaka Nakashima, Kentaro Miyazaki
Nov 12, 2013·Applied and Environmental Microbiology·Nobutaka NakashimaTomohiro Tamura
Oct 31, 2015·Biotechnology Advances·Gert PetersMarjan De Mey
Mar 14, 2013·Letters in Applied Microbiology·N Nakashima, T Tamura
Jun 2, 2015·Biotechnology Advances·Amit Kumar ChaudharyEun Yeol Lee
Dec 21, 2013·RNA Biology·Jorge Vazquez-Anderson, Lydia M Contreras
Aug 18, 2018·PLoS Computational Biology·Gert PetersMarjan De Mey
Apr 13, 2021·Biochemical and Biophysical Research Communications·Shouta FujitaMitsuoki Kawano
Jan 20, 2012·ACS Synthetic Biology·Vandana SharmaYohei Yokobayashi

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