Initiation of Escherichia coli ribosomes on matrix coupled mRNAs studied by optical biosensor technique

The optical biosensor technique, based on the surface plasmon resonance (SP R) phenomenon, has been used to study the initiation of protein synthesis b y E. coli ribosomes on surface coupled mRNA. mRNA was first periodate oxidi zed and then hydrazide coupled to the surface of a CM5 sensor chip. The for mation of initiation complexes on the surface coupled mRNA was monitored in real-time with a BIACORE 2000 instrument. Mature 70S*mRNA*fMet-tRNA(Met) i nitiation complexes were assembled on mRNA by sequential introduction of th e 30S and 50S subunits supplemented with appropriate initiation factors and fMet-tRNA(Met). We show that the formation of 70S*mRNA complexes on the su rface coupled mRNA proceeds efficiently only in the presence of tRNA. Moreo ver, 70S*mRNA*fMet-tRNA(Met) complexes formed with fMet-tRNA(Met) are more stable than similar complexes formed with deacylated tRNAs. The efficient f ormation and slow dissociation of mature 70S*mRNA*fMet-tRNA(Met) initiation complexes are most easily explained by the stabilization of the interactio n of the ribosomal subunits by fMet-tRNA(Met). This work demonstrates the f easibility of the BIACORE technique for studying the initiation of protein synthesis. (C) Societe francaise de biochimie et biologie moleculaire / Edi tions scientifiques et medicales Elsevier SAS.