Ranu R S, London I M, Das A, Dasgupta A, Majumdar A, Ralston R, Roy R, Gupta N K
Proc Natl Acad Sci U S A. 1978 Feb;75(2):745-9. doi: 10.1073/pnas.75.2.745.
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-tRNA(f) (Met) binding factor (IF), which forms a ternary complex with Met-tRNA(f) (Met) 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-tRNA(f) (Met) 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-tRNA(f) (Met) 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 Mg(2+) at 0 degrees . 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'-[beta,gamma-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.
网织红细胞及其裂解物中的蛋白质合成受血红素调控。在血红素缺乏时，一种能阻断多肽链起始的血红素调节性翻译抑制剂（HRI）被激活。HRI是一种蛋白激酶（ATP：蛋白质磷酸转移酶，EC 2.7.1.37），它能特异性地使甲硫氨酰 - tRNA(f)（Met）结合因子（IF）的38000道尔顿亚基磷酸化，该因子与甲硫氨酰 - tRNA(f)（Met）和GTP形成三元复合物，这一发现表明HRI的抑制作用涉及IF的磷酸化。我们研究了HRI在蛋白质链起始部分反应中的作用，在这些反应中，另一种起始因子[三元复合物解离因子（TDF）]和AUG可增强IF促进的甲硫氨酰 - tRNA(f)（Met）与40S核糖体亚基的结合。结果表明，极低浓度的HRI就能显著抑制甲硫氨酰 - tRNA(f)（Met）与40S亚基的结合。HRI的抑制作用需要ATP。在这些条件下，HRI仅使IF的38000道尔顿亚基磷酸化。TDF制剂不仅能促进三元复合物与40S亚基的结合，还能在0℃、5 mM Mg(2+)存在的情况下促进三元复合物的解离。单独将纯化的IF与低浓度的HRI和ATP预温育，不会显著影响其形成三元复合物的能力；然而，TDF促进的三元复合物解离受到抑制。不可水解的类似物腺苷5'-[β,γ-亚氨基]三磷酸不能替代ATP。这些发现表明，磷酸化导致IF发生构象改变，从而抑制了三元复合物与TDF之间的相互作用，而这种相互作用是三元复合物与40S亚基结合所必需的。
