Abstract
Initiation factor IF2 bound to the 70 S initiation complex with 5'-guanylyl methylenediphosphonate was treated with the cross-linking reagent, dimethyl-3,3'-dithiobispropionimidate. Covalent cross-linking of the factor to ribosomes was demonstrated by stabilization of initiation factor IF2-70S complexes during centrifugation at high salt concentrations. Specific cross-linking of the factor to the 50 S proteins L7/L12 was shown by: (a) co-precipitation of the 50 S proteins L7/L12 by antibodies made against initiation factor IF2 and (b) the appearance of radioactive bands containing [32P]phosphoryl initiation factor IF2 in regions of elevated molecular weight following polyacrylamide/dodecyl sulfate gel electrophoresis. One major band had an apparent molecular weight of 132,000, consistent with its being a dimer containing one copy of L7/L12 (13,000) and initiation factor IF2 (120,000). This cross-linked species was shown to contain [32P]phosphoryl initiation factor IF2 and 35S-labeled L7/L12 in separate experiments. It is concluded that L7/L12, which were shown previously to be required for the binding of the factor, are located at or near the initiation factor IF2 binding site on the 70 S initiation complex.
Highlights
It is concluded that L7/L12, which were shown previously to be required for the binding of the factor, are located at or near the initiation factor IF2 binding site on the 70 S initiation complex
Analysis by sucrose density gradient centrifugation in buffers containing low salt of cross-linked 70 S initiation complexes formed with [ +L7/L12 70 S] particles shows that binding of IF2 and met-tRNA is identical to binding by control untreated 70
Identification of the ribosomal proteins which constitute the 70 S binding site for initiation factor IF2 requires that the factor bind
Summary
Materials -Biochemical compounds were obtained as follows: Gpp(CH,)p from Miles Laboratories; DTBP from Pierce Chemical. L12 -Nonradioactive [ -L7/L12 70 S] ribosomes (4 mg/ml) were incubated with a 6-fold molar excess of purified “5S-labeled L7/L12 in Buffer A for 5 min at 30”. Formation of IF2 ‘70 S Complex and Cross-linking with DTBP - The 70 S ribosomes were exhaustively dialyzed against 50 mM triethanolamine/HCl (pH 8.0), 80 rnM KCl, and 5 mM magnesium acetate (Buffer B). After electrophoresis the gels were stained with Coomassie brilliant blue They were either dried under vacuum and exposed to Kodak No-screen x-ray film for autoradiography or prepared for fluorography [27] with 20% (w/w) 2,5-diphenyloxazole in dimethylsulfoxide, dried, and exposed to Kodak RP. The lyophilized protein was resuspended in 25 ~1 of 30 mM Tris/HCl (pH 7.4), 1.0 M KCl, and 20 mM MgCl, containing 10 mM iodoacetamide and incubated in the dark at room temperature for 30 min. Efficiency and was corrected for 3% crossover of 32P counts
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