Abstract
Homogeneous initiation factor MP forms a stable complex with Met-tRNAf which binds to nitrocellulose filters in the absence of ribosomal subunits. Complex formation is rapid at 0 degrees and the rate of reaction is stimulated 20-fold by GTP when freshly prepared initiation factor MP is used. Under optimal assay conditions, a 1:1:1 stoichiometry for initiation factor MP, GTP, and Met-tRNAf is indicated, based on a molecular weight for initiation factor MP of 180,000. Kinetic analysis of ternary complex formation suggests an ordered reaction sequence with binding of GTP followed by binding of Met-tRNAf. However, binding of GTP appears to produce an unstable state which leads to rapid inactivation of initiation factor MP in the absence of Met-tRNAf. Formation of a stable binary complex of initiation factor MP and Met-tRNAf occurs in the absence of GTP. The binary complex cannot subsequently bind GTP. While storage of initiation factor MP at 0 degrees for several weeks has no effect on the rate or extent of Met-tRNAf binding in the presence of GTP, the rate of binary complex formation is increased 10-fold. The binary and ternary complexes appear to bind to 40 S ribosomal subunits with equal efficiency.
Highlights
Homogeneous initiation factor MP forms a stable complex with Met-tRNA, which binds to nitrocellulose filters in the absence of ribosomal subunits
It is important to distinguish between ternary complex formation and subsequent retention on nitrocellulose filters, with homogeneous IF-MP neither process is affected by Mg2+ concentrations as high as 10 mM
Since substrate inhibition is not observed, a GTP concentration which saturates at all input levels of IF-MP, 10 FM, is used in all binding assays, Exogenous Mga+ is not required and has little effect on ternary complex formation from 0 to 10 mM
Summary
Materials-GTP, dithiothreitol, aurintricarboxylic acid, and N-ethylmaleimide were obtained from Calbiochem. 0.5 M KCI extract of reticulocyte ribosomes as described in the preceding paper [24] This preparation of IF-MP contains no detectable methionyl-tRNA synthetase activity (data not shown). Met-tRNA, binding activity is rapidly inactivated by preincubation of IF-MP with GTP, and IF-MP can form stable binary complexes with Met-tRNA, in the absence of GTP (see “Results”), binding reactions were initiated by the addition of protein to the assay buffer containing these ligands. It is important to distinguish between ternary complex formation and subsequent retention on nitrocellulose filters, with homogeneous IF-MP neither process is affected by Mg2+ concentrations as high as 10 mM Under these assay conditions the extent of GTP dependent Met-tRNA, binding increases linearly as a function of IF-MP over the range 0.1 to 1 pg of IF-MP/SO ~1 of binding assay (Fig. 2).
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