Investigation of mRNA quadruplex formation in Escherichia coli. Wieland, M. & Hartig, J. S Nature protocols, 4(11):1632–40, January, 2009. ![Investigation of mRNA quadruplex formation in Escherichia coli. [link]](https://bibbase.org/img/filetypes/link.svg "link")
Paper doi abstract bibtex The protocol presented here allows for the investigation of the formation of unusual nucleic acid structures in the 5'-untranslated region (UTR) of bacteria by correlating gene expression levels to the in vitro stability of the respective structure. In particular, we describe the introduction of G-quadruplex forming sequences close to the ribosome-binding site (RBS) on the mRNA of a reporter gene and the subsequent read-out of the expression levels. Insertion of a stable secondary structure results in the cloaking of RBS and eventually reduced gene expression levels. The structures and stability of the introduced sequences are further characterized by circular dichroism (CD) spectroscopy and thermal melting experiments. The extent of inhibition is then correlated to the stability of the respective quadruplex structure, allowing judgement of whether factors other than thermodynamic stability affect the formation of a given quadruplex sequence in vivo. Measuring gene expression levels takes 2 d including cloning; CD experiments take 5 hours per experiment.
@article{Wieland2009,
title = {Investigation of {mRNA} quadruplex formation in {Escherichia} coli.},
volume = {4},
issn = {1750-2799},
url = {http://www.ncbi.nlm.nih.gov/pubmed/19876023},
doi = {10.1038/nprot.2009.111},
abstract = {The protocol presented here allows for the investigation of the formation of unusual nucleic acid structures in the 5'-untranslated region (UTR) of bacteria by correlating gene expression levels to the in vitro stability of the respective structure. In particular, we describe the introduction of G-quadruplex forming sequences close to the ribosome-binding site (RBS) on the mRNA of a reporter gene and the subsequent read-out of the expression levels. Insertion of a stable secondary structure results in the cloaking of RBS and eventually reduced gene expression levels. The structures and stability of the introduced sequences are further characterized by circular dichroism (CD) spectroscopy and thermal melting experiments. The extent of inhibition is then correlated to the stability of the respective quadruplex structure, allowing judgement of whether factors other than thermodynamic stability affect the formation of a given quadruplex sequence in vivo. Measuring gene expression levels takes 2 d including cloning; CD experiments take 5 hours per experiment.},
number = {11},
journal = {Nature protocols},
author = {Wieland, Markus and Hartig, Jörg S},
month = jan,
year = {2009},
pmid = {19876023},
keywords = {\#nosource, 5' Untranslated Regions, Bacterial, Bacterial: chemistry, Binding Sites, Circular Dichroism, Cloning, Escherichia coli, Escherichia coli: genetics, G-Quadruplexes, Green Fluorescent Proteins, Green Fluorescent Proteins: analysis, Messenger, Messenger: chemistry, Molecular, Molecular: methods, RNA, RNA Stability, Spectrum Analysis},
pages = {1632--40},
}
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