Abstract
Association constants for tRNA binding to poly(U) programmed ribosomes were assessed under standardized conditions with a single preparation of ribosomes, tRNAs, and elongation factors, respectively, at 15 and 10 mM Mg2+. Association constants were determined by Scatchard plot analysis (the constants are given in units of [10(7)/M] measured at 15 mM Mg2+): the ternary complex Phe-tRNA.elongation factor EF-Tu.GTP (12 +/- 3), Phe-tRNA (1 +/- 0.4), AcPhe-tRNA (0.7 +/- 0.3), and deacylated tRNA(Phe) (0.4 +/- 0.15) bind with decreasing affinity to the A site of poly(U)-programmed ribosomes. tRNA(Phe) (7.2 +/- 0.8) binds to the P site with higher affinity than AcPhe-tRNA (3.7 +/- 1.3). The affinity of the E site for deacylated tRNA(Phe) (1 +/- 0.2) is about the same as that of the A site for AcPhe-tRNA (0.7 +/- 0.3). At lower Mg2+ concentrations the affinity of the E site ligand becomes stronger relative to the affinities of the A site ligands. Phe-tRNA and ternary complexes can occupy the A site at 0 degrees C in the presence of poly(U) even if the P site is free, whereas, as already known, deacylated tRNA or AcPhe-tRNA bind first to the P site of programmed ribosomes. Hill plot analyses of the binding data confirm an allosteric linkage between A and E sites in the sense of a negative cooperativity.
Highlights
We present a comprehensive list of apparent associaprogrammed ribosomeswere assessed under standard- tion constants for binding tRNAs to A, P, and E sites
The binding sites of poly(U)-programmed ribosomes were titrated with tRNAs at 10 and 15 mM M e at 0 and 37 "C
Occupation of the E site prevents an occupation of the A site at 0 "C, which has been observed when AcPhe-tRNA was used as an A site ligand [3]
Summary
The binding sites of poly(U)-programmed ribosomes were titrated with tRNAs at 10 and 15 mM M e at 0 and 37 "C. At higher Phe-tRNA amounts the P sites of this minor ribosome population was probably filled with deacylated tRNAPhe,which waspresent in the Phe-tRNA preparation as a contaminant. This interpretatiown as confirmed by thin-layer analysis, which revealedthat themajority of the bound Phe material was present asmono-Phe (65-82%),most of the remaining material being Phez, significant. When P and E sites were occupiedwith deacylated tRNAPhe, the nonenzymatic binding of Phe-tRNA to theA site at 37 "C gave practically the same results asthe corresponding binding after prefilling only the P site. Open circles in G show binding controls at 0 'C
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