Poly (dT) Inhibition of Globin Synthesis in the Rabbit Reticulocyte Lysate System. Reversal of the Inhibition by Poly (dT)-Binding Protein
1982 Volume 91 Issue 6 Pages 1981-1994
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1982 Volume 91 Issue 6 Pages 1981-1994
The mechanism of inhibition of globin synthesis by poly (dT) was studied in the rabbit reticulocyte lysate system. When the lysate was incubated with [14C] poly (dT), poly (dT) was found to bind with the native 40 S ribosomal subunit and the “supernatant factor.” But the binding to the native 40 S ribosomal subunits was not directly related to the poly (dT) inhibition.
Ribosomal subunits were prepared from rabbit reticulocytes and tested for their binding with poly (dT) and their effect on the poly (dT) inhibition. Poly (dT) was found to bind with the derived 40 S ribosomal subunit, but not with the derived 60 S subunit, and the poly (dT) inhibition was slightly reversed by the derived 40 S ribosomal subunit.
Under conditions such that the elongation of nascent chains was inhibited by sparsomycin, the formation of the 80 S/Met-tRNAf complex was inhibited by poly (dT) and the inhibition was greater at high concentration of KOAc. However, the formation of the 40 S/Met-tRNAf complex was inhibited to the same extent at 70mM and 200mM KOAc in the presence of GMPPCP.
A factor (TF) that reverses the poly (dT) inhibition was partially purified from the KCl-wash of rabbit reticulocyte ribosomes by ammonium sulfate fractionation, and Sephadex G-150 and DEAF-cellulose column chromatographies. From the Sephadex G-150 column chromatography of TF, the molecular weight of TF was estimated to be 81, 000-102, 000. TF reversed the poly (dT) inhibition of 80 S/[3H]-mRNA/Met-tRNAf complex or that of 40 S/[3H] mRNA/Met-tRNAf complex. TF bound to [14C] poly (dT) or 3H-labeled globin mRNA. SDS/polyacrylamide slab gel electrophoresis of the complexes between the factor and [14C] poly (dT) or [3H]-mRNA showed common polypeptide bands of 22, 500, 25, 000, and 49, 000 daltons.
These data can be explained by assuming that poly (dT) binds to a factor which is required for the binding of 40 S/Met-tRNAf complex with mRNA to form inactive complexes, and thus inhibits globin synthesis. The relationship between the poly (dT)-binding protein and known initiation factors is discussed.
