The ribosomal E site at low Mg2+: coordinate inactivation of ribosomal functions at Mg2+ concentrations below 10 mM and its prevention by polyamines.
Under standard conditions (Mg2+/150 mM NH4+) ribosomes can quantitatively participate in tRNA binding at Mg2+ concentrations of 12 to 15 mM. The overall poly(U)-directed Phe incorporation and the extent of tRNA binding to either P, E or A sites decrease in a parallel manner when the Mg2+ concentration is lowered below 10 mM. At 4 mM the inactivation amounts to about 80%. The coordinate inactivation of all three binding sites is accompanied by an increasing impairment of the ability to translocate A-site bound AcPhe-tRNA to the P site. The translocation efficiency is already reduced at 10 mM Mg2+, and is completely blocked at 6-8 mM. The severe inactivation seen at 6 mM Mg2+ vanishes when the polyamines spermine (0.6 mM) and spermidine (0.4 mM) are present in the assay; tRNA binding again becomes quantitative, the total Phe synthesis even exceeds that observed in the absence of polyamines by a factor of 4. In the presence of polyamines and low Mg2+ (3 and 6 mM) two essential features of the allosteric three-site model (Rheinberger and Nierhaus, J. Biol. Chem. 261, 9133 (1986] are demonstrated. 1) Deacylated tRNA is not released from the P site, but moves to the E site during the course of translocation. 2) Occupation of the E site reduces the A site affinity and vice versa (allosteric interactions between E and A sites). The quality of an in vitro system for protein synthesis can be assessed by two criteria. First, the incubation conditions must allow a near quantitative tRNA binding. Secondly, protein synthesis should proceed with near in vivo rate and accuracy. The 3 mM Mg2+/NH4+/polyamine-system seems to be the best compromise at present between these two requirements.
Rheinberger
H J
HJ
Max-Planck-Institut für Molekulare Genetik, Abt. Wittmann, Berlin-Dahlem, West Germany.
Nierhaus
K H
KH
D000494:Allosteric Regulation; D001665:Binding Sites; D002474:Cell-Free System; D004926:Escherichia coli / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology*; D008954:Models, Biological; D009994:Osmolar Concentration; D010452:Peptide Biosynthesis; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects*; D010455:Peptides; D010649:Phenylalanine / Q000378:metabolism; D011072:Poly U / Q000235:genetics; D011073:Polyamines / Q000494:pharmacology*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects* / Q000648:ultrastructure
D010455:Peptides; D011073:Polyamines; D012346:RNA, Transfer, Amino Acyl; D011072:Poly U; C012001:polyphenylalanine; D010649:Phenylalanine; D008274:Magnesium