Abstract
Translation of capped and uncapped eukaryotic mRNAs is stimulated by addition of eIF-4B to an mRNA-dependent reticulocyte lysate system. m7G5 ppp inhibits translation of capped but not uncapped mRNAs and reduces translation of capped vaccinia mRNA to the level obtained with uncapped vaccinia mRNA. Exogenous eIF-4B but no other initiation factor reverses inhibition of protein synthesis by m7G5'ppp. Both capped and uncapped mRNAs interact directly with eIF-4B to form a stable complex, which can be detected by a simple nitrocellulose filter binding assay. However, addition of a 5'-cap to beta-eliminated globin mRNA or satellite tobacco necrosis virus RNA (normally uncapped) increased binding affinity of these mRNAs for eIF-4B and causes binding of these mRNAs to become sensitive to inhibition by m7G5'ppp. These results indicate that the role of the mRNA 5'-cap in translation is related specifically to the function of eIF-4B in forming a complex with mRNA (prior to association of mRNA with the 40 S ribosomal subunit) and that both cap and non-cap sequences participate in this process.
Highlights
From the SDepartments ofMedicine and Cell Biology and the Liver Research Center, Albert Einstein College of Medicine, Bronx, Neul Yorh 10461, the #Department of Virology, The Weizmann Institute of Science, Rehocot, Israel, the 11Virus
Translation of capped and uncapped eukaryotic mRNAs is stimulated by addition of eIF-4B to an mRNA-dependent reticulocyte lysate system. m7G”ppp inhibits translation of capped but not uncapped mRNAs and reduces translation of capped vaccinia mRNA to the level obtained with uncapped vaccinia mRNA
It has been proposed that double-stranded loops in the initiation or preinitiation region of mRNA are involved in attachment of mRNA to the ribosome [34,35] and that specific initiation factors might participate in this process
Summary
Materials-All glassware and solutions were freshly prepared and autoclaved prior to use. Conditions for the amount of nuclease and time, which gave low incorporation of labeled amino acids into protein in the absence of added mRNA but high activity in the presence of added mRNA (as close as possible to the level of activity with untreated lysate), were used for bulk preparation of mRNA-dependent reticulocyte lysate This material was stored in small aliquots in liquid nitrogen. Binding of Labeled mRNAs to eIF-4B-Incubations were performed for 5 min at 23°C in a 50.~1 reaction mixture and contained 20 mu Tris-HCl (pH 7.4), 100 mM KCl, 1 mu dithiothreitol, and amounts of purified eIF-4B and labeled RNA as noted in the appropriate table or figure legend. Complex formation was monitored by dilution of the reaction mixture with 3 ml of cold 10 mu Tris-HCl (pH 7.4), 100 mu KC1 and collection and washing of the labeled complex on a nitrocellulose fiter (0.45-p pore size, HAWP, 25-mm diameter, Millipore Corp.) as previously reported [15]
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