The Experts below are selected from a list of 47574 Experts worldwide ranked by ideXlab platform
Kalpana Chakraburtty - One of the best experts on this subject based on the ideXlab platform.
-
Elongation Factor 3 in fungal translation
eLS, 2001Co-Authors: Kalpana ChakraburttyAbstract:Elongation Factor 3 (EF-3) is uniquely required by the fungal translational apparatus. Understanding its function may provide new insights into the mechanistic aspect of translation for all systems. The information in turn may lead to the development of potential antifungal drugs. Keywords: protein synthesis; fungal protein; ribosome; ATP-binding protein; RNA-binding protein
-
evolutionary divergence of an Elongation Factor 3 from cryptococcus neoformans
Journal of Bacteriology, 2001Co-Authors: Greg Blakely, Kalpana Chakraburtty, James Hekman, Peter R WilliamsonAbstract:Elongation Factor 3 (EF3) is considered a promising drug target for the control of fungal diseases because of its requirement for protein synthesis and survival of fungi and a lack of EF3 in the mammalian host. However, EF3 has been characterized only in ascomycete yeast. In order to understand the role of EF3 in a basidiomycete yeast, we cloned the gene encoding EF3 from Cryptococcus neoformans (CnEF3), an important fungal pathogen in immunocompromised patients, including those infected with human immunodeficiency virus. CnEF3 was found to encode a 1,055-amino-acid protein and has 44% identity with EF3 from Saccharomyces cerevisiae (YEF3). Expressed CnEF3 exhibited ATPase activity that was only modestly stimulated by ribosomes from S. cerevisiae. In contrast, CnEF3 showed tight binding to cryptococcal ribosomes, as shown by an inability to be removed under conditions which successfully remove Saccharomyces EF3 from ribosomes (0.5 M KCl or 2 M LiCl). CnEF3 also poorly complemented a YEF3 defect in a diploid null mutant and two temperature-sensitive mutants which have been shown previously to be complemented well by EF3 from other ascomycetes, such as Candida albicans. These data clearly identify the presence of a functioning EF3 in the basidiomycete yeast C. neoformans, which demonstrates an evolutionary divergence from EF3 of ascomycete yeast.
-
limited proteolysis of yeast Elongation Factor 3 sequence and location of the subdomains
Journal of Biological Chemistry, 2000Co-Authors: Ravi Kambampati, Carla Pellegrino, Anthony Paiva, Lee Huang, Liane M Mendemueller, Kalpana ChakraburttyAbstract:Elongation Factor 3 (EF-3) is an ATPase essential for polypeptide chain synthesis in a variety of yeasts and fungi. We used limited proteolysis to study the organization of the subdomains of EF-3. Trypsinolysis of EF-3 at 30 °C resulted in the formation of three fragments with estimated molecular masses of 90, 70, and 50 kDa. Yeast ribosomes protected EF-3 and the large fragments from further degradation. ATP exposed a new tryptic cleavage site and stabilized the 70- and 50-kDa fragments. The conformation of EF-3 as measured by fluorescence spectroscopy did not change upon ATP binding. Poly(G) stimulated proteolysis and quenched the intrinsic fluorescence of EF-3. Using gel mobility shift, we demonstrated a direct interaction between EF-3 and tRNA. Neither tRNA nor rRNA altered the tryptic cleavage pattern. The proteolytic products were sequenced by mass spectrometric analysis. EF-3 is blocked NH2-terminally by an acetylated serine. The 90-, 70-, and 50-kDa fragments are also blocked NH2-terminally, confirming their origin. The 50-kDa fragment (Ser2-Lys443) is the most stable domain in EF-3 with no known function. The 70-kDa fragment (Ser2-Lys668) containing the first nucleotide-binding sequence motif forms the core ATP binding subdomain within the 90-kDa domain. The primary ribosome binding site is located near the loosely structured carboxyl-terminal end.
-
functional interaction of yeast Elongation Factor 3 with yeast ribosomes
The International Journal of Biochemistry & Cell Biology, 1999Co-Authors: Kalpana ChakraburttyAbstract:Abstract Elongation Factor 3 (EF-3) is a unique and essential requirement of the fungal translational apparatus. EF-3 is a monomeric protein with a molecular mass of 116,000. EF-3 is required by yeast ribosomes for in vitro translation and for in vivo growth. The protein stimulates the binding of EF-1α:GTP:aa-tRNA ternary complex to the ribosomal A-site by facilitating release of deacylated-tRNA from the E-site. The reaction requires ATP hydrolysis. EF-3 contains two ATP-binding sequence motifs (NBS). NBSI is sufficient for the intrinsic ATPase function. NBSII is essential for ribosome-stimulated activity. By limited proteolysis, EF-3 was divided into two distinct functional domains. The N-terminal domain lacking the highly charged lysine blocks failed to bind ribosomes and was inactive in the ribosome-stimulated ATPase activity. The C-terminally derived lysine-rich fragment showed strong binding to yeast ribosomes. The purported S5 homology region of EF-3 at the N-terminal end has been reported to interact with 18S ribosomal RNA. We postulate that EF-3 contacts rRNA and/or protein(s) through the C-terminal end. Removal of these residues severely weakens its interaction mediated possibly through the N-terminal domain of the protein.
-
yeast Elongation Factor 3 structure and function
Biological Chemistry, 1998Co-Authors: Kalpana Chakraburtty, Francisco TrianaalonsoAbstract:Elongation Factor 3 (EF-3) is a unique and essential requirement of the fungal translational apparatus. EF-3 is a single polypeptide protein with a molecular weight of 116,000 required by yeast ribosomes for in vitro translation and for in vivo growth. The YEF3 gene, located on chromosome xii, is essential for the survival of yeast. The deduced amino acid sequence of EF-3 has revealed the presence of duplicated ATP-binding cassettes similar to those present in the membrane associated transporters. The carboxy-terminus of EF-3 contains blocks of lysine boxes essential for its functional interaction with yeast ribosomes. EF-3 stimulates binding of aminoacyl-tRNA to the ribosomal A-site by facilitating release of deacylated tRNA from the exit site (E-site). Chasing experiments revealed that EF-3 enhances the rate of tRNA dissociation from the E-site by a Factor of two without affecting the affinity of the site for tRNA. EF-3 function is dependent on ATP hydrolysis. The existence of functional homologs of EF-3 in higher eukaryotes is still an open question. Further investigations are needed to settle this issue.
Mick F Tuite - One of the best experts on this subject based on the ideXlab platform.
-
translation Elongation Factor 3 ef 3 an evolving eukaryotic ribosomal protein
Journal of Molecular Evolution, 1995Co-Authors: Graham Belfield, Natalie Rosssmith, Mick F TuiteAbstract:Fungi appear to be unique in their requirement for a third soluble translation Elongation Factor. This Factor, designated Elongation Factor 3 (EF-3), exhibits ribosome-dependent ATPase and GTPase activities that are not intrinsic to the fungal ribosome but are nevertheless essential for translation Elongation in vivo. The EF-3 polypeptide has been identified in a wide range of fungal species and the gene encoding EF-3 (YEF3) has been isolated from four fungal species (Saccharomyces cerevisiae, Candida albicans, Candida guillermondii, and Pneumocystis carinii). Computer-assisted analysis of the predicted S. cerevisiae EF-3 amino acid sequence was used to identify several potential functional domains; two ATP binding/catalytic domains conserved with equivalent domains in members of the ATP-Binding Cassette (ABC) family of proteins, an amino-terminal region showing significant similarity to the E. coli S5 ribosomal protein, and regions of predicted interaction with rRNA, tRNA, and mRNA. Furthermore, EF-3 was also found to display amino acid similarity to myosin proteins whose cellular function is to provide the motive force of muscle. The identification of these regions provides clues to both the evolution and function of EF-3. The predicted functional regions are conserved among all known fungal EF-3 proteins and a recently described homologue encoded by the Chlorella virus CVK2. We propose that EF-3 may play a role in the ribosomal optimization of the accuracy of fungal protein synthesis by altering the conformation and activity of a ribosomal "accuracy center," which is equivalent to the S4-S5-S12 ribosomal protein accuracy center domain of the E. coli ribosome. Furthermore, we suggest that EF-3 represents an evolving ribosomal protein with properties analogous to the intrinsic ATPase activities of higher eukaryotic ribosomes, which has wider implications for the evolutionary divergence of fungi from other eukaryotes.
-
translation Elongation Factor iii ep 3 an evolving eukaryotic ribosomal protein
1995Co-Authors: Graham Belfield, Natalie Rosssmith, Mick F TuiteAbstract:Fungi appear to be unique in their requirement for a third soluble translation Elongation Factor. This Factor, designated Elongation Factor 3 (EF-3), exhibits ribosome-dependent ATPase and GTPase activities that are not intrinsic to the fungal ribosome but are nevertheless essential for translation Elongation in vivo. The EF-3 polypeptide has been identified in a wide range of fungal species and the gene encoding EF-3 (YEF3) has been isolated from four fungal species (Saccharomyces cerevisiae, Candida albicans, Candida guillermondii, and Pneumocystis carinii). Computer-assisted analysis of the predicted S. cerevisiae EF-3 amino acid sequence was used to identify several potential functional domains; two ATP binding/catalytic domains conserved with equivalent domains in members of the ATP-Binding Cassette (ABC) family of proteins, an amino-terminal region showing significant similarity to the E. coli S5 ribosomal protein, and regions of predicted interaction with rRNA, tRNA, and mRNA. Furthermore, EF-3 was also found to display amino acid similarity to myosin proteins whose cellular function is to provide the motive force of muscle. The identification of these regions provides clues to both the evolution and function of EF-3. The predicted functional regions are conserved among all known fungal EF-3 proteins and a recently described homologue encoded by the Chlorella virus CVK2. We propose that EF-3 may play a role in the ribosomal optimization of the accuracy of fungal protein synthesis by altering the conformation and activity of a ribosomal ''accuracy center,'' which is equivalent to the S4-S5-S12 ribosomal protein accuracy center domain of the E. coli ribosome. Furthermore, we suggest that EF-3 represents an evolving ribosomal protein with properties analogous to the intrinsic ATPase activities of higher eukaryotic ribosomes, which has wider implications for the evolutionary divergence of fungi from other eukaryotes.
-
regulation of the gene encoding translation Elongation Factor 3 during growth and morphogenesis in candida albicans
Microbiology, 1994Co-Authors: Rolf Swoboda, Mick F Tuite, David R Colthurst, Gwyneth Bertram, Neil A R Gow, Graeme Gooday, Alistair J P BrownAbstract:The level of the TEF3 mRNA, which encodes the fungal-specific translation Elongation Factor 3 (EF-3), was measured during the yeast-to-hyphal transition in Candida albicans. In contrast to a previous report, TEF3 mRNA levels were shown to change during dilution into fresh medium, increasing only transiently when dimorphism was induced by either (i) an increase in growth temperature (from 25 degrees C to 37 degrees C) combined with the addition of 10% (v/v) bovine calf serum to the medium, or (ii) an increase in growth temperature (from 25 degrees C to 37 degrees C) combined with an increase in the pH of the medium (from pH 4.5 to 6.5). TEF3 mRNA levels also increased in control cultures under conditions where germ tubes were not formed, but they remained elevated in contrast to cultures undergoing morphological changes. TEF3 mRNA levels were not significantly affected by heat-shock, but were tightly regulated during batch growth of the yeast form, reaching maximal levels in exponential phase. Therefore, the changes in TEF3 expression that accompany the dimorphic transition in C. albicans appear to reflect the underlying physiological changes that occur during morphogenesis and are not a response to morphogenesis per se. For this reason TEF3 mRNA measurement cannot be used as a loading control in Northern analyses of dimorphic gene regulation. Comparison of TEF3 mRNA levels with the abundance of the EF-3 polypeptide indicated that the synthesis of this essential translation Factor might be subject to post-transcriptional regulation.
-
translation Elongation Factor 3 a fungus specific translation Factor
Molecular Microbiology, 1993Co-Authors: Graham Belfield, Mick F TuiteAbstract:Fungi appear to be unique in their requirement for a third soluble translation Elongation Factor. This Factor, designated Elongation Factor 3 (EF-3), was first described in the yeast Saccharomyces cerevisiae and has subsequently been identified in a wide range of fungal species including Candida albicans and Schizosaccharomyces pombe. EF-3 exhibits ribosome-dependent ATPase and GTPase activities that are not intrinsic to the fungal ribosome, but which are essential for translation Elongation. Recent studies on the structure of EF-3 from several fungal species have shown that it consists of a repeated domain, with each domain containing the expected putative ATP- and GTP-binding motifs. Overall, EF-3 shows striking amino acid similarity to members of the ATP-binding Cassette (ABC) family of membrane-associated transport proteins although EF-3 is not itself directly membrane-associated. Regions of the EF-3 polypeptide also show structural homology with other translation-associated Factors including aminoacyl-tRNA synthetases and the Escherichia coli ribosomal protein S5. While the precise role of EF-3 in the translation Elongation cycle remains to be defined, recent evidence suggests that it may be involved in optimizing accuracy during mRNA decoding at the ribosomal A site. Furthermore, the essential nature of EF-3 with respect to the fungal cell indicates that it may be an effective antifungal target. Its apparently ubiquitous occurrence throughout the fungal kingdom also suggests that it may be a useful fungal taxonomic marker.
-
Elongation Factor 3 ef 3 from candida albicans shows both structural and functional similarity to ef 3 from saccharomyces cerevisiae
Molecular Microbiology, 1992Co-Authors: David R Colthurst, B S Schauder, M V Hayes, Mick F TuiteAbstract:As with many other fungi, including the budding yeast Saccharomyces cerevisiae, the dimorphic fungus Candida albicans encodes the novel translation Factor, Elongation Factor 3 (EF-3). Using a rapid affinity chromatography protocol, EF-3 was purified to homogeneity from C. albicans and shown to have an apparent molecular mass of 128 kDa. A polyclonal antibody raised against C. albicans EF-3 also showed cross-reactivity with EF-3 from S. cerevisiae. Similarly, the S. cerevisiae TEF3 gene (encoding EF-3) showed cross-hybridization with genomic DNA from C. albicans in Southern hybridization analysis, demonstrating the existence of a single gene closely related to TEF3 in the C. albicans genome. This gene was cloned by using a 0.7 kb polymerase chain reaction-amplified DNA fragment to screen to C. albicans gene library. DNA sequence analysis of 200 bp of the cloned fragment demonstrated an open reading frame showing 51% predicted amino acid identity between the putative C. albicans EF-3 gene and its S. cerevisiae counterpart over the encoded 65-amino-acid stretch. That the cloned C. albicans sequence did indeed encode EF-3 was confirmed by demonstrating its ability to rescue an otherwise non-viable S. cerevisiae tef3:HIS3 null mutant. Thus EF-3 from C. albicans shows both structural and functional similarity to EF-3 from S. cerevisiae.
Delphine Chaduli - One of the best experts on this subject based on the ideXlab platform.
-
One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes
Persoonia, 2015Co-Authors: J.b. Stielow, C.a. Lévesque, K.a. Seifert, Wieland Meyer, L. Irinyi, D. Smits, R. Renfurm, G.j.m. Verkley, M. Groenewald, Delphine ChaduliAbstract:The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1-D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial beta-tubulin II (TUB2); iv) gamma-actin (ACT); v) translation Elongation Factor 1-alpha (TEF1 alpha); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5-6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation Elongation Factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1 alpha. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1 alpha, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail.
Mikio Arisawa - One of the best experts on this subject based on the ideXlab platform.
-
a monoclonal antibody specific for carboxy terminal region of yeast translation Elongation Factor 3 inhibits ribosome activated atpase activity but not intrinsic atpase activity
Iubmb Life, 1996Co-Authors: Masahiro Uritani, Miho Izuta, Atsushi Tabata, Kazutoshi Nakayama, Makoto Iizumi, Mikio ArisawaAbstract:Elongation Factor-3 (EF-3) is an essential translation Elongation Factor specific to yeasts and fungi. When EF-3 interacts with yeast ribosomes, its ATPase activity, which is indispensable for the function of EF-3 in translation, is drastically enhanced. In this study, a monoclonal antibody specific for the carboxy-terminal region inhibited the ribosome-activated ATPase activity of EF-3, while it did not inhibit the intrinsic ATPase activity of EF-3. The results suggest that the carboxy-terminal region of EF-3 is involved in the interaction with yeast ribosomes. The monoclonal antibody also inhibited poly(U)-directed poly(Phe) synthesis, which indicates that the carboxy-terminal region is important for EF-3 to express its function in the polypeptide Elongation cycle.
-
A point mutation within each of two ATP-binding motifs inactivates the functions of Elongation Factor 3
Biochimica et biophysica acta, 1996Co-Authors: Hemiing Yang, Kenji Hamada, Hiromichi Terashima, Miho Izuta, Emi Yamaguchi-sihta, Osamu Kondoh, Hideo Satoh, Masazumi Miyazaki, Mikio Arisawa, Chikara MiyamotoAbstract:We have investigated how point mutations in the two ATP-binding motifs (G(463)PNGCGK(469)ST and G(701)PNGAGK(707)ST) of Elongation Factor 3 (EF-3) affect ribosome-activated ATPase activity of EF-3, polyphenylalanine synthesis, and growth of Saccharomyces cerevisiae. The point mutation impaired the ribosome-activated ATPase activity of EF-3, when glycine(463 and 701) and lysine(469 and 707) were replaced with valine and arginine, respectively. Thus, each glycine and lysine residue in both ATP-binding motifs is indispensable for EF-3's binding with ATP and the ensuing generation of ribosome-activated ATPase activity. Additionally, the mutant EF-3s did not catalyze polyphenylalanine synthesis in vitro when each glycine(463 and 701) was replaced with valine. The mutant EF-3s did not support cell growth in TEF3-disrupted S. cerevisiae, when each lysine(469 and 707) and glycine(463) was replaced with arginine and valine, respectively. Thus, each of the two ATP-binding motifs of EF-3 is indispensable for the ribosome-activated ATPase activity of EF-3, which is required for protein synthesis and cell growth in S. cerevisiae.
-
polyamino acids that inhibit the interaction of yeast translational Elongation Factor 3 ef 3 with ribosomes
Journal of Biochemistry, 1994Co-Authors: Masahiro Uritani, Kyoko Nakano, Yuhko Aoki, Hisao Shimada, Mikio ArisawaAbstract:EF-3 is a translational Elongation Factor specific to yeasts and fungi. Its carboxy-terminal region contains three lysine-clusters and is very basic. The region has been reported to be responsible for the interaction with ribosomes [Ishiyama, A., Ogawa, K., & Miyazaki, M. (1992) in Abstracts of the 15th Annual Meeting of the Molecular Biology Society of Japan, p.190]. To find specific inhibitors for the interaction of EF-3 with ribosomes, the effects of two basic polyamino acids, poly-L-(Lys) and poly-L-(Arg), and two acidic polyamino acids, poly-L-(Asp) and poly-L-(Glu), were examined using two assay systems for ATPase of EF-3. One was for the ribosome-activated ATPase and the other for the intrinsic (ribosome-independent) ATPase of EF-3. Basic polyamino acids were expected to act as analogues of the carboxy-terminal region of EF-3, and acidic ones to interact with EF-3. The basic polyamino acids inhibited the ribosome-activated ATPase, but they also inhibited the intrinsic one more effectively. Acidic polyamino acids, poly-L-(Asp) and poly-L-(Glu), inhibited the ribosome-activated ATPase but not the intrinsic one. Thus, acidic polyamino acids could be specific inhibitors of the interaction between EF-3 and ribosomes. Furthermore, a system for detecting the binding of EF-3 to ribosomes was constructed. That is, ribosome-bound EF-3 was detected by measuring the ATPase on precipitated ribosomes after a mixture of EF-3 and ribosomes had been ultracentrifuged. Using this system, poly-L-(Asp) was shown to inhibit the binding of EF-3 to ribosomes directly.
Masahiro Uritani - One of the best experts on this subject based on the ideXlab platform.
-
detection and analysis of translation Elongation Factor 3 genes from various yeasts
Bioscience Biotechnology and Biochemistry, 1999Co-Authors: Masahiro Uritani, Yasuhito Shoumura, Shinpei YamadaAbstract:Yeast translation requires a unique Elongation Factor, EF-3. However, information about EF-3 genes has been limited to only a few yeast species. Here, we developed a PCR-based system to detect the EF-3 genes specifically, and identified EF-3 gene fragments from various yeast species in which EF-3 genes have not yet been found.
-
a monoclonal antibody specific for carboxy terminal region of yeast translation Elongation Factor 3 inhibits ribosome activated atpase activity but not intrinsic atpase activity
Iubmb Life, 1996Co-Authors: Masahiro Uritani, Miho Izuta, Atsushi Tabata, Kazutoshi Nakayama, Makoto Iizumi, Mikio ArisawaAbstract:Elongation Factor-3 (EF-3) is an essential translation Elongation Factor specific to yeasts and fungi. When EF-3 interacts with yeast ribosomes, its ATPase activity, which is indispensable for the function of EF-3 in translation, is drastically enhanced. In this study, a monoclonal antibody specific for the carboxy-terminal region inhibited the ribosome-activated ATPase activity of EF-3, while it did not inhibit the intrinsic ATPase activity of EF-3. The results suggest that the carboxy-terminal region of EF-3 is involved in the interaction with yeast ribosomes. The monoclonal antibody also inhibited poly(U)-directed poly(Phe) synthesis, which indicates that the carboxy-terminal region is important for EF-3 to express its function in the polypeptide Elongation cycle.
-
polyamino acids that inhibit the interaction of yeast translational Elongation Factor 3 ef 3 with ribosomes
Journal of Biochemistry, 1994Co-Authors: Masahiro Uritani, Kyoko Nakano, Yuhko Aoki, Hisao Shimada, Mikio ArisawaAbstract:EF-3 is a translational Elongation Factor specific to yeasts and fungi. Its carboxy-terminal region contains three lysine-clusters and is very basic. The region has been reported to be responsible for the interaction with ribosomes [Ishiyama, A., Ogawa, K., & Miyazaki, M. (1992) in Abstracts of the 15th Annual Meeting of the Molecular Biology Society of Japan, p.190]. To find specific inhibitors for the interaction of EF-3 with ribosomes, the effects of two basic polyamino acids, poly-L-(Lys) and poly-L-(Arg), and two acidic polyamino acids, poly-L-(Asp) and poly-L-(Glu), were examined using two assay systems for ATPase of EF-3. One was for the ribosome-activated ATPase and the other for the intrinsic (ribosome-independent) ATPase of EF-3. Basic polyamino acids were expected to act as analogues of the carboxy-terminal region of EF-3, and acidic ones to interact with EF-3. The basic polyamino acids inhibited the ribosome-activated ATPase, but they also inhibited the intrinsic one more effectively. Acidic polyamino acids, poly-L-(Asp) and poly-L-(Glu), inhibited the ribosome-activated ATPase but not the intrinsic one. Thus, acidic polyamino acids could be specific inhibitors of the interaction between EF-3 and ribosomes. Furthermore, a system for detecting the binding of EF-3 to ribosomes was constructed. That is, ribosome-bound EF-3 was detected by measuring the ATPase on precipitated ribosomes after a mixture of EF-3 and ribosomes had been ultracentrifuged. Using this system, poly-L-(Asp) was shown to inhibit the binding of EF-3 to ribosomes directly.