Role of GTP in Eukaryotic Polypeptide‐Chain Initiation

Abstract

1.A factor, which makes a ternary complex with GTP and eukaryotic initiator tRNA (MettRNAi), has been purified 100-fold from developed cysts of Artemia salina. Some of the properties of the purified factor have been studied.2. M 2 + appears to inhibit ternary complex formation. 3. Little or no ternary complex is formed when 5 pM GTP is replaced by an identical concentration of UTP, CTP or ATP. The analog, guanosine 5'-(fi,y-imino)triphosphate [GMP-P(NH)P] seems to be a much better substitute for GTP than guanosine 5'-(B~-methylene)triphosphate [GMP-P(CH2)PI. Since GMP-P(NH)P is as effective as GTP in ternary complex formation, it would appear that GTP plays the role of an allosteric effector in this step of eukaryotic polypeptide chain initiation.4. GDP inhibits both the rate and extent of ternary complex formation. The inhibition is largely reversed by adding a 5-fold molar excess of GTP over GDP. dGDP is slightly less inhibitory than GDP. UDP and CDP are much less inhibitory than GDP and very little inhibition is obtained with ADP.5. The preformed ternary complex is rapidly and completely destroyed in the presence of N-ethylmaleimide. The results suggest that free -SH groups of the factor may be essential for maintaining the integrity of the ternary complex.Despite much work on the mechanism of polypeptide chain initiation in eukaryotic cells, relatively little information is presently available regarding the precise role of GTP in this process. For example, our understanding of the step(s) in which GTP plays an allosteric role, of the step(s) in which GTP is hydrolyzed and of the role of GTP hydrolysis in eukaryotic polypeptide chain initiation, is either incomplete or lacking. In order to clarify the above problems, we have recently undertaken a project directed toward the purification and detailed examination of the properties of eukaryotic protein factor(s) whose functioning in the process of polypeptide chain initiation is either dependent on and/or modified by guanine nucleotides. In the present paper, a procedure is described for 100-fold purification of such a factor from developed cysts of the brine shrimp, Artemiu Ahhrrviations. GMP-P(CH2)P and GMP-P(NH)P, guanosine S'-(p,.;-methylene)triphosphate and guanosine S'-(p,y-imino)triphosphate, respectively; IF-2, prokaryotic initiation factor 2; MalNEt, N-ethylmaleimide, -~ ~ salina. The purified factor forms a ternary complex with GTP and initiator Met-tRNA (Met-tRNAi) of eukaryotic origin. The role of GTP in this reaction appears to be that of an allosteric effector since the analog GMP-P(NH)P is as effective as GTP in ternary complex formation. GMP-P(CH2)P seems to be much less active than GMP-P(NH)P. Although GDP inhibits ternary complex formation, the inhibition is, reversed by increasing concentrations of GTP. We have also observed that the preformed factor . GTP . MettRNAi ternary complex is rapidly destroyed in the presence of N-ethylmaleimide. These and other properties of the purified faFtor are described in this paper.Previous work from other l...

Regulation of the First Step of the Initiation of Brain Protein Synthesis by Guanosine Diphosphate

“…Of the nonhydrolyzable analogs of GTP only GMP-P(NH)P could replace GTP and was, indeed, more effective, while GMP-P(CH,)P was only 15% as effective. This is in agreement with the results of Ochiai-Yanagi and Mazumder (1976) who used initiation factor from A . salina, although a number of others have reported that GMP-P(CH,)P could substitute for GTP to some degree in other systems (Dettman and Stanley, 1972;Levin et al, 1973;Walton and Gill, 1975;Harbitz and Hauge, 1976).…”

“…When GDP is present in equimolar concentration with GTP, ternary complex formation is inhibited 32% with a purified preparation of initiation factor from A . salina (Ochiai-Yanagi and Mazumder, 1976), 56% in the crude KC1 wash fraction from Ehrlich ascites tumor cells (Smith and Henshaw, 1975), and 60% and 70% in a postribosomal supernatant from rat skeletal and heart muscle, respectively (Rannels et al, 1976). This is to be compared with an 85% inhibition in our brain system when GDP and GTP are added in equimolar amounts (results not shown).…”