The Binding of Tritiated Elongation Factors 1 and 2 to Ribosomes from Krebs II Mouse Ascites Tumor Cells

Nolan, Robert D.; Grasmuk, Hans; Drews, Jürgen

doi:10.1111/j.1432-1033.1975.tb09815.x

Cited by 55 publications

(28 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It was, therefore, inferred from earlier experiments, that the EF-1 and EF-2 binding sites on the ribosome at least overlap even if they are not identical [2-41. Further apparent confirmation of this conclusion was derived from initial experiments where binding of [3H]EF-1 and ['4C]Phe-tRNA to poly(U)-programmed ribosomes was significantly inhibited, when the ribosomes had been preincubated with EF-2 and GMP-P(CH2)P [5].…”

Section: Discussionsupporting

confidence: 54%

See 1 more Smart Citation

Further Evidence that Elongation Factor 1 Remains Bound to Ribosomes during Peptide Chain Elongation

Grasmuk

Nolan

Drews

1977

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

This paper describes three types of experiments which indicate that the binding sites for elongation factor 1 (EF-1) and elongation factor 2 (EF-2) on ascites cell ribosomes are not identical and perhaps not even overlapping. The experimental evidence presented includes direct competitive binding of labeled elongation factors to ribosomes as well as the influence of pokeweed antiviral protein and Escherichia coli anti-L7/L12 proteins on the binding and function of the two factors. It is further shown that EF-1P from Artemia salina does not function in displacing EF-1 from mouse ascites tumor cell ribosomes.These results also support our recently proposed model that EF-1 remains bound to the ribosome during the peptide chain elongation cycle.

show abstract

Section: Discussionsupporting

confidence: 54%

“…The radioactivity of the fractions was determined by liquid scintillation counting. Any radioactivity found eluting in the ribosome region was taken to be ribosome-associated labeled elongation factor [5].…”

Section: Analytical Techniquesmentioning

confidence: 99%

Further Evidence that Elongation Factor 1 Remains Bound to Ribosomes during Peptide Chain Elongation

Grasmuk

Nolan

Drews

1977

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…ribosome interaction [7,36,371. In order to identify the guanosine-binding domain, we used the guanosine nucleotide analogue oGTP as an affinity label.…”

Section: Sites Of Adp-ribosylation and Gtp Bindingmentioning

confidence: 99%

Localization of the sites of ADP‐ribosylation and GTP binding in the eukaryotic elongation factor EF‐2

Nilsson

Nygård

1985

European Journal of Biochemistry

View full text Add to dashboard Cite

Tryptic cleavage of EF-2, molecular mass 93 kDa, produced an 82-kDa polypeptide and a 10-kDa fragment, which was further degraded. By a slower reaction the 82-kDa polypeptide was gradually split into a 48-kDa and a 34-kDa fragment. Similarly, treatment with chymotrypsin resulted in the formation of an 82-kDa polypeptide and a small fragment. In contrast to the tryptic 82-kDa polypeptide the corresponding chymotryptic cleavage product was relatively resistant to further attack. The degradation of the 82-kDa polypeptide with either trypsin or chymotrypsin was facilitated by the presence of guanosine nucleotides, indicating a conformational shift in native EF-2 upon nucleotide binding. No effect was observed in the presence of ATP, indicating that the effect was specific for guanosine nucleotides. After affinity labelling of native EF-2 with oxidized [3H]GTP and subsequent trypsin treatment the radioactivity was recovered in the 48-kDa polypeptide showing that the GTPbinding site was located within this part of the factor. Correspondingly, tryptic degradation of EF-2 labelled with [I4C]NAD+ in the presence of diphtheria toxin showed that the site of ADP-ribosylation was within the 34-kDa polypeptide. By cleavage with the tryptophan-specific reagent N-chlorosuccinimide the site of ADP-ribosylation could be located at a distance of 40-60 kDa from the GTP-binding site and about 4-11 kDa from the nearest terminus.The eukaryotic elongation factor EF-2 consists of a single polypeptide chain with a molecular mass of 93 kDa [l]. The factor catalyses the translocation of peptidyl-tRNA from the ribosomal aminoacyl site to the peptidyl site [2 -41. EF-2 forms stoichiometric, binary complexes with guanosine nucleotides [5, 61, and the association of the factor with ribosomes is strictly dependent on the nucleotide [7]. The translocation of peptidyl-tRNA is coupled to a ribosome and EF-Zdependent GTP hydrolysis [8, 91, leading to a reduced affinity of the factor for the ribosome [lo]. As in the case of the prokaryotic factor EF-G [l 1 -131 the GTP-cleaving center seems to be located in the factor [14]. Thus, the factor may have at least three functional domains, i. e. those involved in GTP and ribosome binding and the catalytic center responsible for GTP hydrolysis. The domains are interdependent since (a) GTP binding is strictly required for the association of the factor with the ribosome [7, 81 and (b) this interaction induces the GTP hydrolysis [8, 91. EF-2 is ADP-ribosylated in the presence of NAD+ and diphtheria toxin [15]. Although the GTP-binding capacity i s unaffected [16,17], the presence of GTP inhibits the modification of the factor [17,18]. The protective effect of GTP seems to be related to a conformational change in the factor. The ADP-ribosylation is accompanied by a reduced affinity for the ribosome [lo] ultimately leading to a decreased GTP hydrolysis [18, 191 and an inhibition of the translation process Correspondence to 0. Nygird, Avd. for cellfysiologi, WennerGrens Institut, Stockholms universitet, Nor...

show abstract

“…The ribosomal solutions thus obtained (1 9 ml/tube) was layered onto a discontinuous sucrose gradient consisting of 8 ml of 1 M sucrose and 7 ml of 0.4 M sucrose both containing 0.3 M KC1, 3 mM MgC12, 10 mM 2-mercaptoethanol and 20 mM Tris/HCl, pH 7.6. The gradient was centrifuged in a Hitachi 50T angle rotor at 50000 rpm for 16 h. The pellet was suspended in a buffer consisting of 0.2 M sucrose, 0.1 M NH,Cl, 5 mM MgCI2, 0.5 mM dithiothreitol, and 20 mM Tris/HCl, pH 7.6 at a concentration of 300 Az60 units/ml and stored at -80°C as 80s ribosomes. The 80s ribosomes obtained had no detectable EF-2 activity when examined for polyphenylalanine synthesis according to Iwasaki and Kaziro [lo].…”

mentioning

confidence: 99%

Cross-linking of elongation factor 2 to rat-liver ribosomal proteins by 2-iminothiolane

et al. 1986

View full text Add to dashboard Cite

Complexes containing rat liver 80s ribosomes treated with puromycin and high concentrations of KCl, elongation factor 2 (EF-2) from pig liver, and guanosine 5'-[/3,y-methylene]triphosphate were prepared. Neighboring proteins in the complexes were cross-linked with the bifunctional reagent 2-iminothiolane. Proteins were extracted and then separated into 22 fractions by chromatography on carboxymethylcellulose of which seven fractions were used for further analyses. Each protein fraction was subjected to diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Nine cross-linked protein pairs between EF-2 and ribosomal proteins were shifted from the line formed with monomeric proteins. The spots of ribosomal proteins cross-linked to EF-2 were cut out from the gel plate and labelled with lZ5I. The labelled protein was extracted from the gel and identified by three kinds of two-dimensional gel electrophoresis, followed by autoradiography. The following proteins of both large and small subunits were identified: L9, L12, L23, LA33 (acidic protein of M, 33000), P2, S6 and S23/ S24, and L3 and L4 in lower yields. The results are discussed in relation to the topographies of ribosomal proteins in large and small subunits. Furthermore we found new neighboring protein pairs in large subunits, LA33 -L11 and LA33 -L12.During protein biosynthesis in eukaryotic cells, elongation factor 2 (EF-2) interacts with ribosomes in the presence of GTP and catalyzes the translocation of newly synthesized peptidyl-tRNA from the aminoacyl-tRNA binding site (the A site) of ribosomes to the peptidyl-tRNA site (the P site) [l -31. The functional roles of ribosomal proteins in the translocation are as yet unknown. To understand the molecular mechanisms of the translocation in mammalian ribosomes, it is important to identify proteins in the EF-2 binding site of ribosomes.Employing four kinds of cross-linking reagents and hydrogen peroxide, we identified 62 cross-linked protein pairs involving 36 protein species containing two acidic proteins of rat liver 60s subunits and have discussed these in relation to other functional data [4, 51. Concerning the 40s subunits, similar data were presented by Tolan and Traut [6]. Therefore, it is of interest that ribosomal proteins which interact with elongation factors have been determined.In the present experiments we investigated EF-2-binding sites in rat liver 80s ribosomes, employing 2-iminothiolane. Cross-linked proteins were analyzed by a modified method of diagonal SDS gel electrophoresis [7]. Seven large-subunit proteins and two small-subunit proteins were identified as the components cross-linked to EF-2. The mutual locations of these proteins in the 80s ribosomes are discussed.

show abstract

The Binding of Tritiated Elongation Factors 1 and 2 to Ribosomes from Krebs II Mouse Ascites Tumor Cells

Cited by 55 publications

References 27 publications

Further Evidence that Elongation Factor 1 Remains Bound to Ribosomes during Peptide Chain Elongation

Further Evidence that Elongation Factor 1 Remains Bound to Ribosomes during Peptide Chain Elongation

Localization of the sites of ADP‐ribosylation and GTP binding in the eukaryotic elongation factor EF‐2

Cross-linking of elongation factor 2 to rat-liver ribosomal proteins by 2-iminothiolane

Contact Info

Product

Resources

About