期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 112, 期 44, 页码 E6038-E6047出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1518958112
关键词
quorum sensing; regulatory sRNA; Qrr; RSort-Seq; regulation
资金
- Howard Hughes Medical Institute
- National Science Foundation [MCB-0343821]
- Defense Advanced Research Projects Agency Biochronicity Program [D12AP00025]
- W. M. Keck Foundation
- NIH [F32AI085922, 5R01GM065859]
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [0948112] Funding Source: National Science Foundation
Five homologous noncoding small RNAs (sRNAs), called the Qrr1-5 sRNAs, function in the Vibrio harveyi quorum-sensing cascade to drive its operation. Qrr1-5 use four different regulatory mechanisms to control the expression of similar to 20 mRNA targets. Little is known about the roles individual nucleotides play in mRNA target selection, in determining regulatory mechanism, or in defining Qrr potency and dynamics of target regulation. To identify the nucleotides vital for Qrr function, we developed a method we call RSort-Seq that combines saturating mutagenesis, fluorescence-activated cell sorting, high-throughput sequencing, and mutual information theory to explore the role that every nucleotide in Qrr4 plays in regulation of two mRNA targets, luxR and luxO. Companion biochemical assays allowed us to assign specific regulatory functions/underlying molecular mechanisms to each important base. This strategy yielded a regional map of nucleotides in Qrr4 vital for stability, Hfq interaction, stem-loop formation, and base pairing to both luxR and luxO, to luxR only, and to luxO only. In terms of nucleotides critical for sRNA function, the RSort-Seq analysis provided strikingly different results from those predicted by commonly used regulatory RNA-folding algorithms. This approach is applicable to any RNA-RNA interaction, including sRNAs in other bacteria and regulatory RNAs in higher organisms.
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