ABSTRACT
Chemical probing is often used to gain knowledge on the secondary and tertiary structures of RNA molecules either free or engaged in complexes with ligands. The method monitors the reactivity of each nucleotide towards chemicals of various specificities reflecting the hydrogen bonding environment of each nucleotide within the RNA molecule. In addition, information can be obtained on the binding site of a ligand (noncoding RNAs, protein, metabolites), and on RNA conformational changes that accompanied ligand binding or perturbation of the environmental cues. The detection of the modifications can be obtained either by using end-labeled RNA molecules or by primer extension using reverse transcriptase. The goal of this chapter is to provide the reader with an experimental guide to probe the structure of RNA in vitro and in vivo with the most suitable chemical probes.
Subject(s)
Computational Biology/methods , Nucleic Acid Conformation , RNA/chemistry , Electrophoresis, Polyacrylamide Gel , Models, Molecular , RNA Cleavage , RNA FoldingABSTRACT
We have adapted a method to map cell surface proteins and to monitor the effect of specific regulatory RNAs on the surface composition of the bacteria. This method involves direct labeling of surface proteins of living bacteria using fluorescent dyes and a subsequent separation of the crude extract by 2D gel electrophoresis. The strategy yields a substantial enrichment in surface proteins over cytoplasmic proteins. We validated this method by monitoring the effect of the regulatory RNA MicA in Escherichia coli, which regulates the synthesis of several outer membrane proteins, and highlighted the role of Staphylococcus aureus RNAIII for the maintenance of cell wall integrity.
Subject(s)
Bacterial Proteins/metabolism , Cell Membrane/metabolism , Escherichia coli/metabolism , RNA, Bacterial/metabolism , Staphylococcus aureus/metabolism , Bacterial Outer Membrane Proteins/metabolism , Base Sequence , Carbocyanines/metabolism , Cell Wall/metabolism , Electrophoresis, Gel, Two-Dimensional/methods , Escherichia coli/genetics , Microscopy, Confocal , Molecular Sequence Data , RNA, Bacterial/genetics , Reproducibility of Results , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Staining and Labeling/methods , Staphylococcus aureus/geneticsABSTRACT
Staphylococcus aureus is one of the major human pathogens, which causes numerous community-associated and hospital-acquired infections. The regulation of the expression of numerous virulence factors is coordinated by complex interplays between two component systems, transcriptional regulatory proteins, and regulatory RNAs. Recent studies have identified numerous novel RNAs comprising cis-acting regulatory RNAs, antisense RNAs, small non coding RNAs and small mRNAs encoding peptides. We present here several examples of RNAs regulating S. aureus pathogenicity and describe various aspects of antisense regulation.