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. 1978 Feb;75(2):745–749. doi: 10.1073/pnas.75.2.745

Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase: Inhibition of interaction of Met-tRNAfMet binding factor with another initiation factor in formation of Met-tRNAfMet·40S ribosomal subunit complexes

R S Ranu *, I M London *,†,‡, A Das §, A Dasgupta §, A Majumdar §, R Ralston §, R Roy §, N K Gupta §
PMCID: PMC411333  PMID: 273238

Abstract

Protein synthesis in reticulocytes and their lysates is regulated by heme. In heme deficiency a heme-regulated translational inhibitor (HRI) that blocks initiation of polypeptide chains is activated. HRI is a protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNAfMet binding factor (IF), which forms a ternary complex with Met-tRNAfMet and GTP, a finding that suggests that the inhibition by HRI involves the phosphorylation of IF.

We have investigated the effect of HRI in the partial reactions of protein chain initiation in which the IF-promoted binding of Met-tRNAfMet to 40S ribosomal subunits is enhanced by another initiation factor [ternary complex dissociation factor (TDF)] and AUG. The results show that HRI at very low concentrations markedly inhibits the binding of Met-tRNAfMet to 40S subunits. The inhibitory effect of HRI requires ATP. Under these conditions HRI phosphorylates only the 38,000-dalton subunit of IF.

The TDF preparations not only promote the binding of the ternary complex to 40S subunits but also promote the dissociation of the ternary complex in the presence of 5 mM Mg2+ at 0°. The preincubation of purified IF alone with low concentrations of HRI and ATP does not significantly affect its capacity to form the ternary complex; however, the TDF-promoted dissociation of the ternary complex is inhibited. The nonhydrolyzable analog adenosine 5′-[β,γ-imido]triphosphate does not substitute for ATP. These findings suggest that phosphorylation causes a conformational modification in IF, which results in inhibition of the interaction between the ternary complex and TDF that is required for the binding of the ternary complex to 40S subunits.

Keywords: phosphorylation of Met-tRNAfMet binding factor, translational regulation, inhibition of Met-tRNAfMet binding, 40S ribosomal subunits, protein—protein interaction

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Selected References

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