Apparent association constants of tRNAs for the ribosomal A, P, and E sites.
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.
Schilling-Bartetzko
S
S
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin-Dahlem, Germany.
Franceschi
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F
Sternbach
H
H
Nierhaus
K H
KH
D000215:Acylation; D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent; D004926:Escherichia coli / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D011072:Poly U / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D012343:RNA, Transfer / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
D002413:Cations, Divalent; D012333:RNA, Messenger; D012360:RNA, Transfer, Phe; D011072:Poly U; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D010444:Peptide Elongation Factor Tu; D008274:Magnesium