Mechanism of translocation: effect of cognate transfer ribonucleic acids on the binding of AUGUn to 70S ribosomes

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Abstract
The unidirectional sliding-type movement of the PP-tRNA.cntdot.mRNA complex from Escherichia coli with respect to the ribosome was studied by looking at the effect of different combinations of cognate tRNA on the stability of the 70S.cntdot.AUGUn complex. The association constant for the binary complex 70S.cntdot.AUGU3 was determined as 6.8 .times. 105 M-1. Addition of .**GRAPHIC**. resulted in a 67-fold increase in the association constant, which with both cognate tRNA is revised to Kassoc = 2.2 .times. 108 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.cntdot.mRNA interaction is governed by the presence of the cognate tRNA 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, one concludes that the affinity of the peptidyl transfer center for the peptidyl moiety pulls the PP-tRNA.cntdot.mRNA complex from the A (aminoacyl-tRNA) site to the P (peptidyl-tRNA) site. EF-G.cntdot.GTP or EF-G.cntdot.GMPPCP 5''-(.beta.,.gamma.-methylene)triphosphate] displace .**GRAPHIC**. from the quaternary complex 70S.cntdot.AUGUn.cntdot. .**GRAPHIC**. .cntdot.tRNAPhe (n = 3 and 6) at Mg2+ < 25 mM. From the amount of EF-G.cntdot.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.cntdot.GTP-dependent release of the deacylated tRNA from the P site, followed by removal of EF-G.cntdot.GDP from the 50S subunit, is sufficient to trigger the translocation of the mRNA.cntdot.PP-tRNA complex.