Mechanism of translocation: effect of cognate transfer ribonucleic acids on the binding of AUGUn to 70S ribosomes.
We try to mimic the unidirectional sliding-type movement of the PP-tRNA . mRNA complex with respect to the ribosome by looking at the effect of different combinations of cognate tRNAs on the stability of the 70S-AUGUn complex. The association constant for the binary complex 70S-AUGU3 was determined as 6.8 x 10(5) M-1. Addition of tRNAfMet resulted in a 67-fold increase in the association constant, which with both cognate tRNAs is revised to Kassoc = 2.2 x 10(8) M-1. Increasing the chain length of the oligonucleotide from AUGU3 to AUGU13 did not further raise the association constant. The data indicate that the stability of the 70S ribosome . mRNA interaction is governed by the presence of the cognate tRNAs and is topographically restricted to the decoding domains. Since a peptidyl group in the tRNA increases the affinity of AUGU3 for the ribosome by up to 15-fold, we conclude that the affinity of the peptidyl transfer center for the peptidyl moiety pulls the PP-tRNA . mRNA complex from the A (aminoacyl-tRNA) site to the P (peptidyl-tRNA) site. EF-G . GTP or EF-G . GMPPCP 5'-(beta, gamma-methylene)triphosphate] displace tRNAfMet from the quaternary complex 70S . AUGUn . tRNAfMet . tRNAPhe (n = 3 and 6) at Mg2+ less than 25 mM. From the amount of EF-G . GTP bound to a 70S ribosome, it follows that the elongation factor replaces the deacylated tRNA in a stoichiometric way. These data indicate that the EF-G . GTP-dependent release of the deacylated tRNA from the P site, followed by removal of EF-G . GDP from the 50S subunit, is sufficient to trigger the translocation of the mRNA . PP-tRNA complex.
D001692:Biological Transport; D004926:Escherichia coli / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009841:Oligonucleotides / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000378:metabolism*; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D009841:Oligonucleotides; D009843:Oligoribonucleotides; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D012333:RNA, Messenger; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D008274:Magnesium