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Massive functional mapping of a 5'-UTR by saturation mutagenesis, phenotypic sorting and deep sequencing
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
2013 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 12, e122- p.Article in journal (Refereed) Published
Abstract [en]

We present here a method that enables functional screening of large number of mutations in a single experiment through the combination of random mutagenesis, phenotypic cell sorting and high-throughput sequencing. As a test case, we studied post-transcriptional gene regulation of the bacterial csgD messenger RNA, which is regulated by a small RNA (sRNA). A 109 bp sequence within the csgD 5'-UTR, containing all elements for expression and sRNA-dependent control, was mutagenized close to saturation. We monitored expression from a translational gfp fusion and collected fractions of cells with distinct expression levels by fluorescence-activated cell sorting. Deep sequencing of mutant plasmids from cells in different activity-sorted fractions identified functionally important positions in the messenger RNA that impact on intrinsic (translational activity per se) and extrinsic (sRNA-based) gene regulation. The results obtained corroborate previously published data. In addition to pinpointing nucleotide positions that change expression levels, our approach also reveals mutations that are silent in terms of gene expression and/or regulation. This method provides a simple and informative tool for studies of regulatory sequences in RNA, in particular addressing RNA structure-function relationships (e.g. sRNA-mediated control, riboswitch elements). However, slight protocol modifications also permit mapping of functional DNA elements and functionally important regions in proteins.

Place, publisher, year, edition, pages
2013. Vol. 41, no 12, e122- p.
National Category
Natural Sciences Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-206462DOI: 10.1093/nar/gkt267ISI: 000321057100003OAI: oai:DiVA.org:uu-206462DiVA: diva2:644649
Available from: 2013-09-01 Created: 2013-08-30 Last updated: 2017-12-06Bibliographically approved

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