Tritiated elongation factors 1 and 2 (EF-1 and EF-2) were obtained from Krebs I1 ascites cells which had been grown in mice injected with radioactive amino acids. The highly purified factors were sufficiently radioactive to be used in a study of the interactions between ribosomes and elongation factors.The following results were obtained. 1. EF-1 binding to ribosomes requires the presence of a polynucleotide, an aminoacyl-tRNA specified by the latter and a guanosine nucleotide carrying three phosphate groups. The hydrolysis of the GTP molecule involved in the binding reaction leads to the immediate release of EF-1. If GTP is replaced by Guo-5'-P,-CH2-P the factor remains bound to the ribosome and can be detected by sucrose gradient centrifugation techniques.2. Likewise EF-2 binding to ribosomes can only be detected in the presence of Guo-5'-P2-CH2-P. 3. The affinity of ribosomes for EF-2 appears to be higher than for EF-1: preincubation of ribosomes with EF-2 inhibits the subsequent attachment of EF-1 almost completely. EF-1 prebound to ribosomes in the presence of Guo-5'-P2-CH2-P, poly(uridy1ic acid) and Phe-tRNAPhe is partially removed from the ribosomes together with Phe-tRNA during a second incubation with EF-2.4. Although EF-2 binding to ribosomes precludes any stable association between ribosomes and EF-1 it does not prevent the insertion of aminoacyl-tRNA into the ribosomal A-site. The attachment of aminoacyl-tRNA under these conditions enhances the binding of EF-2 to the ribosome.5. The antibiotic showdomycin strongly inhibits the attachment of EF-1 to ribosomes and to a lesser degree impairs the binding of EF-2.6. A-site ribosomes display a strong preference for the attachment of EF-2 and bind EF-1 only very poorly. The reverse is true for P-site ribosomes which are good substrates for the binding of EF-1 and bind EF-2 less efficiently than A-site ribosomes.These results and a number of additional findings made in this and in previous studies are discussed in the general context of the structure and function of mammalian elongation factors 1 and 2.Elongation factor 1 (EF-1) from ascites tumor cells occurs in multiple forms. All of these appear to be composed of aggregates of different numbers of a single polypeptide chain with an approximate molecular weight of 47000 [l]. The pure tetramer form of EF-1 was shown to carry one binding site for guanosine nucleotides which can be occupied by GTP, GDP or Guo-S-P,-CH,-P [2]. The binding constants of EF-1 for both GDP and GTP appear to be approximately lo5 M-' but only the binding of GTP conveyedAbbreviations. EF-1, elongation factor 1 ; EF-2, elongation factor 2; Guo-5'-P2-CH2-P, 5'-guanylyl-methylene-diphosphonate.to elongation factor 1 the capacity to specifically interact with the aminoacyl-tRNA [2]. The fact that Guo-5'-CH2-P could promote the EF-I-directed binding of aminoacyl-tRNA to ribosomes [3], while only promoting slight aminoacyl-tRNA protection against deacylation in an EF-1-dependent protection assay [2], indicated that these two functions of th...

Cytidine 5'-triphosphate:cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) was purified 2,300-fold from frozen Escherichia coli B cells. The enzyme catalyzed the formation of CMP-KDO, a very labile product, from CTP and KDO. No other sugar tested could replace KDO as an alternate substrate. Uridine 5'-triphosphate at pH 9.5 and deoxycytidine 5'-triphosphate at pH 8.0 and 9.5 could be used as alternate substrates in place of CTP. CMP-KDO synthetase required Mg2+ at a concentration of 10.0 mM for optimal activity. The pH optimum was determined to be between 9.6 and 9.3 in tris(hydroxymethyl)aminomethane-acetate or sodium-glycine buffer. This enzyme had an isoelectric point between pH 4.15 and 4.4 and appeared to be a single polypeptide chain with a molecular weight of 36,000 to 40,000. The apparent Km values for CTP and KDO in the presence of 10.0 mM Mg2+ were determined to be 2.0 X 10(-4) and 2.9 X 10(-4) M, respectively, at pH 9.5. Uridine 5'-triphosphate and deoxycytidine 5'-triphosphate had apparent Km values of 8.8 X 10(-4) and 3.4 X 10(-4) M. respectively, at pH 9.5.

The effect of a number of different antibiotics and toxins on the capacity of Krebs II mouse ascites ribosomes to bind 3H‐labelled elongation factors (EF‐1 and EF‐2) has been examined. It was found that abrin and ricin inhibit the binding of EF‐2, while diphtheria toxin, sparsomycin, streptovitacin A, and cycloheximide had essentially no effect on its binding. Of the other compounds examined, sparsomycin was unique in its capacity, under some circumstances, to significantly affect the binding of aminoacyl‐tRNA and EF‐1 to ribosomes. Fusidic acid appears to nonspecifically enhance the binding of both EF‐1 and EF‐2.

An entirely new model for the mechanism of elongation factor 1 (EF-1) function is presented. Experiments, in which mixtures of rH]EF-1, ribosomes from Krebs I1 ascites cells and various additional co-factors were analyzed by chromatography on Sepharose 6B, show that EF-1 binds to the ribosome early in the translation process and remains bound on the ribosome during translation. Optimal EF-1 binding occurs on polynucleotide-programmed ribosomes carrying a tRNA in their P-site.On the other hand it was clearly shown that EF-2 attached at each translocation event and was then released before a new Phe-tRNA could be bound.

A procedure for the purification of elongation factor 1 (EF-1) from KrebsII mouse ascites cells is described. Ascites EF-1 occurs in multiple forms of different molecular weight like the corresponding enzymes from calf brain and rat liver. Biogel A-5m chromatography of our purest EF-1 resulted in a pattern indicating the presence of different molecular weight components. However, only a single polypeptide band was observed when this same material was analysed by electrophoresis on acrylamide gels containing sodium dodecylsulphate. With this method the molecular weight of the EF-1 polypeptide chain was determined to be 47 000. It is suggested that the occurrence of multiple forms of EF-1 is due to the association of 47000-mol. wt subunits to form aggregates of different sizes.The factor has essentially the same enzymatic properties which were described for EF-1 from ret,iculocytes, calf brain and rat liver. The attachment of aminoacyl-tRNA to ribosomes catalyzed by EF-1 follows a strict 1 : 1 stoichiometry. EF-2 when introduced into the ribosomal binding assay allows the recycling of EF-1 and subsequent synthesis of polypeptide chains. The possible implications of this finding with respect to the conditions which allow stoichiometric or catalytic function of EF-1 are discussed.Elongation factor 1 functions in polypeptide synthesis by catalyzing the attachment of aminoacyl-tRNA to the ribosomal acceptor site [l, 21. This reaction has been shown to require GTP and some evidence has recently been produced suggesting the formation of a ternary complex between aminoacyl-tRNA, GTP and EF-1 as a necessary intermediate in the binding of aminoacyl-tRNA to the ribosomal A-site [3,4]. It has also been shown that the interaction of the aminoacyl-tRNA anticodon with the complementary codon is a prerequisite for the cleavage of those GTP molecules which became attached to the ribosomal A-site as part of this ternary complex [3]. Apart from this specific GTP hydrolysis into GDP and phosphate, an uncoupled GTPase activity is displayed by EF-1 in the presence of ribosomes and arninoacyl-tRNA [5,6], the functional significance of which is not known.I n spite of the advances which have recently been made in understanding the function of EF-1, many questions pertaining to the structure as well as the function of this protein remain to be resolved.