The Role of Eucaryotic Elongation Factor Tu in Protein Synthesis

Abstract

A sensitive radioimmunoassay for eucaryotic elongation factor Tu (eEF-Tu) was developed using radioiodinated elongation factor T (eEF-T) and goat anti-(rabbit eEF-T) immunoglobulins coupled to a solid support. eEF-T was iodinated with lZ5I to a specific activity of 7 x lo3 counts min-' ng-' using a system employing lactoperoxidase and glucose oxidase coupled to a solid support. The assay exhibits a limit of detection of about 1 ng eEF-Tu and an intraassay variability of < 10 %. By using the radioimmunoassay, it was found that eEF-Tu is a major non-hemoglobin protein of rabbit reticulocyte postribosomal supernatant proteins, comprising about 3 of the total hemoglobin and 10-13 % of the non-hemoglobin proteins. Similar results were found for a number of different tissues and cells, including rabbit heart, brain, liver and kidneys, as well as both induced and non induced Friend erythroleukemia cells. Values of eEF-Tu ranged from 1 % of supernatant proteins in heart to about 11 %, in noninduced erythroleukemic cells. The levels of eEF-Tu in these mammalian tissues were comparable to the level of the homologous factor EF-Tu in Escherichia coli. It has previously been found that EF-Tu constitutes about 6 -8 % of the supernatant proteins of E. coli [Furano, A. V. (1975) Proc. Natl Acad. Sci. USA, 72, 4780-47841. The level of eEF-Tu in reticulocytes was compared to the abundance of other components of protein synthesis in reticulocytes, such as translocase (eEF-G), tRNA, ribosomes and eIF-2. In all cases eEF-Tu was present in large excess over these other components.

Translational repression of mRNA for eucaryotic elongation factors in Friend erythroleukemia cells

“…In the case of eEF-Tu, work in this laboratory has shown that the factor is a major constituent of animal cells, including FEL cells [20]. This finding has been confirmed recently for HeLa cells [26] and is supported by our observation that eEF-Tu is an abundant translation product of FEL cell polysomal mRNA (Fig.…”

“…Elongation factors eEF-T, eEF-Tu and eEF-Ts were purified from rabbit reticulocytes by published methods [19, 201. Goat anti-(rabbit eEF-T) antibodies [20] were purified by immunoaffinity chromatography using columns of eEF-T coupled to Sepharose 4B. Oligo (dT)-cellulose was obtained from Bethesda Research Laboratories Inc.…”

Eukaryotic elongation factor Tu is present in mRNA‐protein complexes

“…Preparation of rabbit reticulocyte lysate, UV crosslinking, and [14C]-formaldehyde labeling was done as described [10]. eEF-Tu was purified as in [11]. 2D gel analysis of mRNP proteins by non-equilibrium pH gradient electrophoresis in the first dimension and SDS-Volume 224, number 1…”

“…On the basis of these data it seems likely that mRNA in rabbit reticulocytes is associated with eEF-Tu. We further investigated the presence of eEF-Tu in mRNP by probing Western blots of mRNP proteins with an affinity-purified antibody raised in goats against purified rabbit elongation factor 1 [11]. UV-crosslinked mRNP proteins and an eEFTu marker were electrophoretically transferred to nitrocellulose.…”

“…The control antibody was rabbit antibody against human IgG obtained from Worthington-Cappel. The eEF-Tu antibody has been described in [11]. In both cases antibody was affinity-purified by binding to the corresponding immobilized antigen.…”