Real-time dynamics of ribosome-ligand interaction by time-resolved chemical probing methods.
Three protocols to perform time-resolved in situ probing of rRNA are described. The three methods (chemical modification with DMS and rRNA backbone cleavage by hydroxyl radicals generated by either K-peroxonitrite or Fe(II)-EDTA) make use of a quench-flow apparatus and exploit reactions that are faster than the interactions of ribosomal subunits with their ligands. These methods allow the investigation of the path and dynamics, in a approximately equal 50 to 1500ms time range, of the binding and dissociation of ribosomal ligands.
Fabbretti
Attilio
A
Department of Biology MCA, University of Camerino, Camerino, Italy.
Milon
Pohl
P
Giuliodori
Anna Maria
AM
Gualerzi
Claudio O
CO
Pon
Cynthia L
CL
D000477:Alkylating Agents / Q000737:chemistry / Q000378:metabolism; D004492:Edetic Acid / Q000737:chemistry / Q000378:metabolism; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism*; D015203:Reproducibility of Results; D012270:Ribosomes / Q000378:metabolism*; D013463:Sulfuric Acid Esters / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
D000477:Alkylating Agents; D005296:Ferrous Compounds; D008024:Ligands; D015335:Molecular Probes; D012335:RNA, Ribosomal; D013463:Sulfuric Acid Esters; D030421:Peroxynitrous Acid; C055526:Fe(II)-EDTA; D004492:Edetic Acid; C007482:dimethyl sulfate