Initiation of Escherichia coli ribosomes on matrix coupled mRNAs studied by optical biosensor technique.
The optical biosensor technique, based on the surface plasmon resonance (SPR) phenomenon, has been used to study the initiation of protein synthesis by E. coli ribosomes on surface coupled mRNA. mRNA was first periodate oxidized and then hydrazide coupled to the surface of a CM5 sensor chip. The formation of initiation complexes on the surface coupled mRNA was monitored in real-time with a BIACORE 2000 instrument. Mature 70S*mRNA*fMet-tRNA(Met) initiation complexes were assembled on mRNA by sequential introduction of the 30S and 50S subunits supplemented with appropriate initiation factors and fMet-tRNA(Met). We show that the formation of 70S*mRNA complexes on the surface coupled mRNA proceeds efficiently only in the presence of tRNA. Moreover, 70S*mRNA*fMet-tRNA(Met) complexes formed with fMet-tRNA(Met) are more stable than similar complexes formed with deacylated tRNAs. The efficient formation and slow dissociation of mature 70S*mRNA*fMet-tRNA(Met) initiation complexes are most easily explained by the stabilization of the interaction of the ribosomal subunits by fMet-tRNA(Met). This work demonstrates the feasibility of the BIACORE technique for studying the initiation of protein synthesis.
Karlsson
M
M
Department of Cell and Molecular Biology, BMC, Box 596, 75124 Uppsala, Sweden.
Pavlov
M Y
MY
Malmqvist
M
M
Persson
B
B
Ehrenberg
M
M
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D010442:Peptide Chain Initiation, Translational; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D012329:RNA, Bacterial; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; C089238:fMet-tRNA(fMet)