| Popis: |
An assay was developed to detect the component which recognizes the methylated 5'-terminus of messenger RNA (cap) during initiation of translation. Globin mRNA translation in a reticulocyte cell-free system was partially inhibited with cap analogues, and protein fractions were added to the system in an attempt to reverse inhibition. Such an activity was detected in the 500 mM KCl extract of rabbit reticulocyte ribosomes. The activity (cap analogue inhibition reversal) was purified 800-fold by precipitation with ammonium sulfate saturation, batchwise chromatography on DEAE-cellulose, centrifugation on sucrose gradients containing 100 and 500 mM KCl, and column chromatography on DEAE-cellulose and phosphocellulose. At some stages multiple peaks of activity were detected. Electrophoretic analysis of the final preparation revealed a single polypeptide of 24,000 daltons, making it likely that it is the same as the cap-binding protein detected by Sonenberg et al. (Sonenberg, N., Morgan, M. A., Merrick, W. C., and Shatkin, A. J. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 4843-4847), using a cross-linking assay. Direct binding of purified fractions of cap analog inhibition reversal factor to capped oligonucleotides of the form m7Gppp(Np)6-8G[32P]Cp could be demonstrated by both gel filtration on Sephadex G-50 and nitrocellulose membrane filtration. Binding was stimulated by MgCl2 and nucleoside triphosphates. An approximate association constant between oligonucleotide and protein of 3 X 10(8) M-1 was obtained. |
