A. Majumdar

Protein synthesis in rabbit reticulocytes. XV: Isolation of a ribosomal protein factor (CO-EIF-1) which stimulates Met-tRNAfMet binding to EIF-1☆

Abstract A protein factor, CO-EIF-1, has been partially purified from the high salt wash of reticulocyte ribosomes. CO-EIF-1, by itself, does not bind Met-tRNA f Met but significantly stimulates (2–3 fold) Met-tRNA f Met binding to EIF-1 and also increases the initial rate of this binding reaction. In the presence of CO-EIF-1, approximately 90 percent of the input EIF-1 (assuming a molecular weight of 140,000) was bound to Met-tRNA f Met .

Protein synthesis in rabbit reticulocytes XIX: EIF-2 promotes dissociation of Met-tRNAf-EIF-1-GTP complex and Met-tRNAf binding to 40S ribosomes.

Abstract The peptide chain initiation factor, EIF-2 has been partially purified from the 0.5 M KCl ribosomal wash. The molecular weight of EIF-2 is approximately 450,000. The purified EIF-2 preparation promotes the dissociation of the ternary complex, Met-tRNA f ·EIF-1·GTP in the presence of Mg ++ and is also required along with EIF-1 for AUG-directed Met-tRNA f binding to 40S ribosomes.

Protein synthesis in rabbit reticulocytes XIII: Lack of mRNA (poly r(A)) binding activity in highly purified EIF-1

Abstract Met-tRNA f Met binding factor (EIF-1) has been purified more than 100 fold over crude high salt (0.5 M KCl) ribosomal wash. The purified factor binds 2 nmoles Met-tRNA f Met per mg protein and shows very little poly r(A) binding activity. Crude ribosomal high salt wash possesses significant amounts of poly r(A) binding activity and also binds to other RNAs. The bulk of this unspecific RNA binding protein is separated from EIF-1 by DEAE-cellulose chromatography.

Protein synthesis initiation in eucaryotic cells: A comparative study of the mechanism of peptide chain initiation using cell-free systems from mouse ascites tumor cells and rabbit reticulocytes

Abstract Peptide chain initiation factors were partially purified from the high salt wash of the ascites tumor cell ribosomes, and the characteristics of peptide chain initiation were compared using the ascites and reticulocytes initiation factors and Met-tRNA f arrange staggered Met from reticulocytes, ascites, and yeast. The results are: (i) The ascites peptide chain initiation factor EIF (eucaryotic initiation factor)-1 binds Met-tRNA f arrange staggered Met in the presence of GTP. The crude initiation factor preparation also contains nonspecific RNA binding protein which also binds [ 35 S]Met-tRNA f arrange staggered Met in the absence of GTP. This nonspecific RNA binding protein is progressively removed during purification of EIF-1. (ii) The ternary complex Met-tRNA f arrange staggered Met · EIF-1 · GTP formed with the partially purified Met-tRNA f arrange staggered Met binding factor dissociates extensively upon addition of Mg 2+ . This dissociation reaction is also observed when the nonhydrolyzable GTP analog GMP-PCP is used in place of GTP. (iii) A Co-EIF-1 activity similar to that described in the reticulocyte system is present in the high salt wash of ascites ribosomes and has been partially purified. Addition of either ascites or reticulocyte Co-EIF-1 stimulates the Met-tRNA f arrange staggered Met binding to both ascites and reticulocyte EIF-1. The ternary complex formed with either reticulocyte or ascites purified EIF-1 breaks down extensively in the presence of 3 × 10 −5 , m aurintricarboxylic acid. However, when both EIF-1 and Co-EIF-1 are present, the complex is almost completely resistant to aurintricarboxylic acid (3 × 10 −5 , m ). (iv) The partially purified ascites peptide chain initiation factor preparation efficiently catalyzes Met-tRNA f arrange staggered Met binding to 40 S ribosomes and such binding is strongly AUG dependent. The Met-tRNA f arrange staggered Met · 40 S · AUG complex upon addition of 60 S ribosome forms an 80 S initiation complex active in methionine-puromycin synthesis at low concentrations of Mg 2+ .

Protein synthesis in rabbit reticulocytes XII: Requirement of mRNA (AUG codon) for Met-tRNAfMet binding to 40S ribosomes*

Summary A Millipore filtration assay method has been developed for studies of codon directed Met-tRNAfMet binding to 40S ribosomes. Under the assay conditions, the stimulation of Met-tRNAfMet binding to Millipore filters by added 40S ribosomes is strongly dependent on the presence of AUG codon, and partially purified peptide chain initiation factors. Crude 0.5 M KC1 ribosomal wash is inactive in catalyzing this binding reaction due to the presence of an inhibitor in this preparation.

Protein synthesis in rabbit reticulocytes: Evidence for two forms of the Met-tRNAfMet binding factors

Summary Two forms of Met-tRNA f Met binding factors (IF1A and IF1B) were seperated by DEAE-cellulose chromatography. These two forms can be distinguished by the stabilities of their respective Met-tRNA f Met :IF1:GTP complexes to Mg ++ . The Met-tRNA f Met :IF1B:GTP complex dissociates extensively in the presence of Mg ++ where as the Met-tRNA f Met :IF1A:GTP complex is distinctly more stable under similar conditions. The molecular weight of Met-tRNA f Met :IF1A:GTP complex as determined by sucrose density gradient centrifugation is 160,000 and that of the Met-tRNA f Met :IF1B:GTP complex is 65,000.