Attachment of the 5'-terminal portion of globin mRNAs to 5S-RNA X L5-protein in the 80S initiation complex

Abstract

An 80S initiation complex was formed by incubating a heterologous cell-free system with 125I-labeled globin mRNAs in the presence of sparsomycin. The 80S initiation complex was then digested with micrococcal nuclease. The ribosomal 5S-RNA X L5-protein (5S RNP) fraction, released by EDTA treatment, contained 125I-labeled mRNA fragments. The attachment of labeled mRNA fragments to 5S RNP was shown by (a) CsCl isopycnic centrifugation, (b) recentrifugation through a sucrose density gradient and (c) acrylamide gel electrophoresis of 5S RNP purified by (b). Labeled fragments were released from 5S RNP by treatment with sodium dodecyl sulfate or pronase, indicating the participation of protein L5 in the attachment. The attached mRNA fragments were 23-25 nucleotides in length. Hybridization experiments, using restriction fragments of cDNA for rabbit beta globin mRNA, showed that the attached mRNA fragments were derived from the 5' portion of globin mRNAs. The attachment of 125I-labeled mRNA fragments to 5S RNP was also observed in the 80S initiation complex formed by incubation of reticulocyte lysate with 125I-labeled globin mRNA, but not in labeled polysomal fractions. These findings may indicate that 5S RNP interacted with the 5' portion of globin mRNA, containing the translation initiation codon of globin mRNA in the 80S initiation complex. The biochemical significance of these results is discussed.