Escherichia coli ribosomal protein L20 binds as a single monomer to its own mRNA bearing two potential binding sites.
Ribosomal protein L20 is crucial for the assembly of the large ribosomal subunit and represses the translation of its own mRNA. L20 mRNA carries two L20-binding sites, the first folding into a pseudoknot and the second into an imperfect stem and loop. These two sites and the L20-binding site on 23S ribosomal RNA are recognized similarly using a single RNA-binding site located on one face of L20. In this work, using gel filtration and fluorescence cross-correlation spectroscopy (FCCS) experiments, we first exclude the possibility that L20 forms a dimer, which would allow each monomer to bind one site of the mRNA. Secondly we show, using affinity purification and FCCS experiments, that only one molecule of L20 binds to the L20 mRNA despite the presence of two potential binding sites. Thirdly, using RNA chemical probing, we show that the two L20-binding sites are in interaction. This interaction provides an explanation for the single occupancy of the mRNA. The two interacting sites could form a single hybrid site or the binding of L20 to a first site may inhibit binding to the second. Models of regulation compatible with our data are discussed.
Allemand
F
F
UPR9073 du CNRS, l'Université de Paris VII, Institut de Biologie Physico-chimique, 13 rue Pierre et Marie Curie, 75005, Paris, France.
Haentjens
J
J
Chiaruttini
C
C
Royer
C
C
Springer
M
M
D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D029968:Escherichia coli Proteins; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C488992:rplT protein, E coli