PROTEIN-SYNTHESIS IN RABBIT RETICULOCYTES .14. CHARACTERISTICS OF MESSENGER-RNA (AUG CODON)-DEPENDENT BINDING OF MET-TRANSFER-RNAFMET TO 40 S AND 80 S RIBOSOMES

  • 1 January 1976
    • journal article
    • research article
    • Vol. 251  (20) , 6379-6387
Abstract
The characteristics of Met-tRNAfMet binding to ribosomes (40 S and 80 S) were studied using a 2-stage assay method and the complexes formed were analyzed either by Millipore filtration or by sucrose density gradient centrifugation. With both assay methods, Met-tRNAfMet binding to 40 S ribosomes was entirely dependent upon addition of a partially purified mixture of initiation factors and AUG codon. This binding occurred over a wide Mg2+ concentration range; significant binding was observed even at 20 mM Mg2+. Upon addition of 60 S ribosomes, a significant part of Met-tRNAfMet bound to 40 S ribosomes was transferred to 80 S ribosomes, a significant part of Met-tRNAfMet bound to 40 S ribosomes was transferred to 80 S complex. This transfer reaction had a sharp Mg2+ optimum around 2 mM. The Met-tRNAfMet .cntdot. 80 S .cntdot. AUG complex formed was active in Met-puromycin synthesis. Met-tRNAfMet deacylase present in crude 0.5 M KCL ribosomal wash is a potent inhibitor of the binding reaction as it deacylates Met-tRNAfMet in the Met-tRNAfMet .cntdot. 40 S .cntdot. AUG complex; Glutaraldehyde (0.5%) degrades Met-tRNAfMet .cntdot. 40 S .cntdot. AUG complex but increases the background binding of Met-tRNAfMet to 40 S ribosomes in the absence of AUG codon. Polynucleotides containing uracil and adenosine are strong inhibitors of Met-tRNAfMet binding to 40 S ribosomes. The order of inhibitory activites of the polynucleotides tested was poly(rU) .cntdot. poly(rA) (2:1) > poly(rU) .cntdot. poly(rA) (1:1) > poly(rU) > poly(rA). Other RNAs tested such as poly(rC), poly(rI) .cntdot. poly(rC) and .vphi.6 bacteriophage RNA (double-stranded) were without significant effects on the Met-tRNAfMet-binding reaction.