Probing mRNA structure and sRNA-mRNA interactions in bacteria using enzymes and lead(II).
Enzymatic probing and lead(II)-induced cleavages have been developed to study the secondary structure of RNA molecules either free or engaged in complex with different ligands. Using a combination of probes with different specificities (unpaired vs. paired regions), it is possible to get information on the accessibility of each nucleotide, on the binding site of a ligand (noncoding RNAs, protein, metabolites), and on RNA conformational changes that accompanied ligand binding or environmental conditions (temperature, pH, ions, etc.). The detection of the cleavages can be conducted by two different ways, which are chosen according to the length of the studied RNA. The first method uses end-labeled RNA molecules and the second one involves primer extension by reverse transcriptase. We provide here an experimental procedure that was designed to map the structure of mRNA and mRNA-sRNA interaction in vitro.
Chevalier
Clément
C
Université de Strasbourg, F-67084, Strasbourg, France.
Geissmann
Thomas
T
Helfer
Anne-Catherine
AC
Romby
Pascale
P
D001483:Base Sequence; D005591:Chemical Fractionation; D004798:Enzymes / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D007854:Lead / Q000494:pharmacology*; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects; D012313:RNA / Q000378:metabolism; D012329:RNA, Bacterial / Q000138:chemical synthesis; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D022661:RNA, Untranslated / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000187:drug effects / Q000378:metabolism*
D004798:Enzymes; C034477:RNA primers; D012329:RNA, Bacterial; D012333:RNA, Messenger; D022661:RNA, Untranslated; D007854:Lead; D012313:RNA