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REGULATION OF PROTEIN SYNTHESIS IN MAMMALIAN CELLS: EFFECT OF AURINTRICARBOXYLIC ACID ON METHIONYL-TRANSFER-RNA(F) : EIF-2 : GTP COMPLEX AND CHARACTERISTICS OF A NUCLEASE TREATED LYSATE SYSTEM
Abstract
(A) Eukaryotic initiation factor (eIF-2) can from a ternary complex with Met-tRNA(,f)('Met) and GTP. The Met-tRNA(,f)('Met):eIF-2:GTP complex can be quantitatively retained on nitrocellulose filters. Its formation is drastically inhibited by very low concentrations of aurintricarboxylic acid (ATA). The observed inhibition by ATA becomes more severe when more homogeneous preparations of eIF-2 are used for complex formation. A factor that has been isolated from the 0.5 M KCl ribosomal wash, termed Co-eIF-2A, can protect the ternary complex from degradation by ATA. Co-eIF-2A can also stimulate the rate of ternary complex formation although it can not substitute for eIF-2 in the complex and can bind Met-tRNA(,f)('Met) independently. Similar results were obtained using factors isolated from an Ehrlich ascites cell free system. (B) The micrococcal nuclease treated rabbit reticulocyte lysate system of Pelham and Jackson (77) is used in order to study the effects of several factors in a complete protein synthesizing system. Because the system possesses little if any endogenous translating ability due to the absence of mRNA, it is ideal for studying factors affecting initiation. A detailed description of the system is presented. The heme-regulated inhibitor (HRI) isolated from the S(,100) preparation from rabbit reticulocytes can block translation almost completely after a short lag period. In addition, a factor called ribosomal rescue factor (rRF) can relieve most of the HRI inhibition in this system. Another inhibitor, activated by incubating lysate with double stranded RNA and ATP (dsI) can greatly inhibit the translation activity of the system. (C) The nuclease treated lysate system described in Section B was used to study the effects of 7-methylguanosine 5'-triphosphate (pppm('7)G) on protein synthesis. At low potassium ion levels, the inhibition by pppm('7)G (cap) is minimal. As the potassium ion concentration in the system is raised, cap inhibition becomes more pronounced. A factor (CF) isolated from the 0.5 M KCl ribosomal wash of rabbit reticulocytes can significantly reverse the inhibition by cap. This factor is sensitive to N-ethylmaleimide and heat and has no Met-tRNA(,f)('Met) binding activity associated with it. An approximate molecular weight as determined by the comparison of standards on glycerol density gradients was found to be 20,000 daltons.
Subject Area
Biochemistry
Recommended Citation
PALMIERI, STEVEN WAYNE, "REGULATION OF PROTEIN SYNTHESIS IN MAMMALIAN CELLS: EFFECT OF AURINTRICARBOXYLIC ACID ON METHIONYL-TRANSFER-RNA(F) : EIF-2 : GTP COMPLEX AND CHARACTERISTICS OF A NUCLEASE TREATED LYSATE SYSTEM" (1981). ETD collection for University of Nebraska-Lincoln. AAI8127161.
https://digitalcommons.unl.edu/dissertations/AAI8127161