Translational repression of the Escherichia coli alpha operon mRNA: importance of an mRNA conformational switch and a ternary entrapment complex.
Ribosomal protein S4 represses synthesis of the four ribosomal proteins (including itself) in the Escherichia coli alpha operon by binding to a nested pseudoknot structure that spans the ribosome binding site. A model for the repression mechanism previously proposed two unusual features: (i) the mRNA switches between conformations that are "active" or "inactive" in translation, with S4 as an allosteric effector of the inactive form, and (ii) S4 holds the 30 S subunit in an unproductive complex on the mRNA ("entrapment"), in contrast to direct competition between repressor and ribosome binding ("displacement"). These two key points have been experimentally tested. First, it is found that the mRNA pseudoknot exists in an equilibrium between two conformers with different electrophoretic mobilities. S4 selectively binds to one form of the RNA, as predicted for an allosteric effector; binding of ribosomal 30 S subunits is nearly equal in the two forms. Second, we have used S4 labeled at a unique cysteine with either of two fluorophores to characterize its interactions with mRNA and 30 S subunits. Equilibrium experiments detect the formation of a specific ternary complex of S4, mRNA pseudoknot, and 30 S subunits. The existence of this ternary complex is unambiguous evidence for translational repression of the alpha operon by an entrapment mechanism.
Schlax
P J
PJ
Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Xavier
K A
KA
Gluick
T C
TC
Draper
D E
DE
D000495:Allosteric Site; D016880:Anisotropy; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003062:Codon; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009876:Operon / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature
D003062:Codon; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C040469:ribosomal protein S30; C031798:ribosomal protein S4