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PROTEIN SYNTHESIS IN RABBIT RETICULOCYTES AND YEAST: REQUIREMENTS FOR MET-TRANSFER-RNA(F).40S PREINITIATION COMPLEX FORMATION WITH AUG-CODON AND PHYSIOLOGICAL MESSENGER-RNAS
Abstract
Under standard conditions, in the presence of GTP, highly purified rabbit reticulocytes eIF-2 and Co-eIF-2 factor preparations efficiently stimulated AUG-codon dependent but not physiological mRNA-dependent Met-tRNA(,f) binding to 40S ribosomes. With mRNA replacement of GTP by a nonhydrolyzable GTP analog, GMP-PNP gave significant stimulation of Met-tRNA(,f) binding to 40S ribosomes dependent on physiological mRNAs. Lower but significant stimulation of Met-tRNA(,f) binding to 40S ribosomes was also observed when GTP was used in the presence of nucleoside 5'-diphosphate kinase (NDK) and ATP. ATP alone in the absence of NDK had no significant effect. Under similar condition, yeast (Saccharomyces cerevisiae) eIF-2(,y) and Co-eIF-2A(,y) failed to stimulate AUG-codon dependent Met-tRNA(,f) binding to 40S ribosomes in the presence of GTP. Replacement of GTP by GMP-PNP gave significant stimulation of AUG-codon dependent but not physiological mRNA dependent Met-tRNA(,f) binding to 40S ribosomes. A protein factor with Co-eIF-2A-like activity purified from 0.5M KCl yeast ribosomal salt wash using anti-Co-eIF-2A(,y)('20)-sepharose column was necessary to form the mRNA dependent Met-tRNA(,f).40S complex. This is the first report on the formation of stable Met-tRNA(,f).40S initiation complexes dependent on physiological mRNA, and the first report of a protein factor requirement for such complex formation in yeast. It is also the first comparison of factor requirements for 40S complex formation from two widely divergent eukaryotic cell, namely rabbit reticulocytes and yeast. Besides the Mr 20,000 polypeptide, another polypeptide (Mr 80,000) with Co-eIF-2A activity has been isolated from 0.5M KCl ribosomal salt wash of yeast using anti-Co-eIF-2A(,y)('20)-sepharose affinity column as an initial step of purification. The Mr 80,000 polypeptide (Co-eIF-2A(,y)('80)) has been purified to homogeneity. Its activities are similar to those of Co-eIF-2A(,y)('20) (Ahmad, M. F., Nasrin, N., Banerjee, A. C., and Gupta, N. K. (1985) J. Biol. Chem. 260, 6955-6959). Co-eIF-2A(,y)('80) stimulated Met-tRNA(,f) binding to eIF-2(,y) and the complex was resistant to aurintricarboxylic acid. This complex was also N-ethylmaleimide resistant and heat labile. Met-tRNA(,f) from the preformed quarternary (Met-tRNA(,f).eIF-2(,y).GTP.Co-eIF-2A(,y)) was more rapidly transferred to 40S ribosomes than without the formation of the quarternary complex.
Subject Area
Biochemistry
Recommended Citation
NASRIN, NARGIS, "PROTEIN SYNTHESIS IN RABBIT RETICULOCYTES AND YEAST: REQUIREMENTS FOR MET-TRANSFER-RNA(F).40S PREINITIATION COMPLEX FORMATION WITH AUG-CODON AND PHYSIOLOGICAL MESSENGER-RNAS" (1985). ETD collection for University of Nebraska-Lincoln. AAI8609808.
https://digitalcommons.unl.edu/dissertations/AAI8609808