gamma-Carboxyglutamic acid in eukaryotic and prokaryotic ribosomes.

γ-carboxyglutamic acid (GLA) has been assayed in ribosomes of wheat germ and E. coli, and in E. coli ribosomal sub-units, by amino acid analysis of total protein. Results, as nMoles GLA/mg protein, were 18.1 (wheat germ), 58.4 (E. coli), 39.6 and 81.5 (E. coli 30S and 50S sub-units, respectively.) Results for wheat germ and previously reported mammalian ribosomes were highly similar. Hence the level of GLA in eukaryotic ribosomes appears to be approximately constant, but low compared to bacterial (E. coli) ribosomes.     

Codon-specific interaction of uncharged transfer-RNA with eukaryotic ribosomes.

Rat liver ribosomes bound [32P]tRNAPhe in both a codon-dependent and codon-independent manner. The codon-dependent binding was studied further by utilising the ability of the unchanged tRNAPhe to inhibit the poly(U)-directed binding of [3H]Phe-tRNA to ribosomes. At least part of the codon-dependent binding of uncharged tRNA appears to be to the ribosomal A-site.     

Euglena gracilis chloroplast ribosomes: improved isolation procedure and comparison of elongation factor specificity with prokaryotic and eukaryotic ribosomes

An improved method for the isolation of Euglena chloroplast ribosomes is described which presents a number of advantages over past procedures. First, ribosomes are prepared from whole cell extracts, thus bypassing the need to isolate intact chloroplasts and resulting in a 10-fold improvement in yield. Second, the inclusion of 40 mm Mg²⁺ in the preparation buffers, while stabilizing the chloroplast ribosomes, precipitates and, thereby, virtually eliminates the cytoplasmic 89 S ribosomes. Third, greater than 95% of the chloroplast ribosomes sediment at 68 S rather than as the damaged 53 S particle frequently generated in other preparation procedures. Fourth, even with a high-salt wash to remove endogenous factors, the chloroplast ribosomes still sediment at 68 S and are just as active in in vitro protein synthesis as are E. coli ribosomes. These ribosomes have been tested for activity with elongation factors from prokaryotes, eukaryotes, and the chloroplast itself, and the results have been compared to those obtained with E. coli and wheat germ ribosomes. The data may be summarized as follows: (a) Chloroplast ribosomes use E. coli$\text{EF}\text{-}\text{Tu}\text{Ts}$ and EF-G with the same efficiency as do E. coli ribosomes in protein synthesis, (b) E. coli and chloroplast ribosomes can use Euglena chloroplast EF-G to catalyze translocation, but wheat germ ribosomes cannot, (c) Wheat germ EF-1_H and EF-2 are highly active in polymerization with wheat germ ribosomes, but ribosomes from neither E. coli nor the chloroplast are able to recognize these factors, (d) All three types of ribosomes accept Phe-tRNA from E. coli EF-Tu although to differing degrees. However, neither chloroplast nor E. coli ribosomes recognize wheat germ EF-1_H for the binding of Phe-tRNA.     

The prokaryotic characteristics of Eimeria tenella ribosomes

• 1.1. Ribosomes were purified from the oocysts of Eimeria tenella and characterized. Those in the unsporulated oocysts were mostly in the form of polysomes which became gradually dissociated during the early phase of sporulation and were mostly in the monomeric form in sporulated oocysts.
• 2.2. The monomeric E. tenella ribosome had the size of about 80S and consisted of 60 and 40S subunits. It was readily and completely dissociated to subunits at high potassium concentrations, and its subunits could not hybridize with those of chicken liver ribosome.
• 3.3. The E. tenella ribosomal RNA was estimated to have sedimentation coefficients of 5, 16 and 23S.
• 4.4. The E. tenella ribosomal proteins had a gel electrophoretic pattern similar to that of E. coli.
• 5.5. The in vitro polypeptide synthesis mediated by E. tenella ribosomes was inhibitable by tetracycline and chloramphenicol which are also active against the parasite in vivo.
• 6.6. Chloramphenicol bound to E. tenella ribosomes with a dissociation constant of about 10⁻⁵ M.
• 7.7. All the evidence demonstrates prokaryotic characteristics of the E. tenella ribosome and suggests that the parasite may be a primitive eukaryote.

     

Physico-chemical characteristics of the new antitumor antibiotic virenomycin

Virenomycin, a new crystalline antitumor antibiotic was isolated from the mycelium of Streptomyces virens. The antibiotic contained: C 64.87 per cent, H 5.66 per cent, methoxylic groups 9.5 per cent. The melting temperature was 255-260 degrees (dec.), [alpha]20D=-17 (c 0.142, chloroform). Virenomycin had a complex UV spectrum with lambdamax. 245 (677), 265 (453), 275 (542), 287 (507), 395 (222) nm. A chromofor fragment and carbohydrate (C7H14O5) were found in the methanolysis products. Virenomycin was close to antibiotic c B-21085 BY THe physico-chemical properties and differed from it in the character of the UV spectrum and the values of the specific absorption, as well as by the optic rotation in dimethyl sulphoxide and acetic acid.     

Structural and functional studies of the interaction of the eukaryotic elongation factor EF-2 with GTP and ribosomes

The structure of the guanosine nucleotide binding site of EF-2 was studied by affinity labelling with the GTP analogue, oxidized GTP (oGTP), and by amino acid sequencing of polypeptides generated after partial degradation with trypsin and N-chlorosuccinimide. Native EF-2 contains two exposed trypsin-sensitive cleavage sites. One site is at Arg66 with a second site at Lys571/Lys572. oGTP was covalently bound to the factor between Arg66 and Lys571. After further cleavage of this fragment with the tryptophan-specific cleavage reagent N-chlorosuccinimide, oGTP was found associated with a polypeptide fragment originating from a cleavage at Trp261 and Trp343. The covalent oGTP . EF-2 complex was capable of forming a high-affinity complex with ribosomes, indicating that oGTP, in this respect, induced a conformation in EF-2 indistinguishable from that produced by GTP. Although GTP could be substituted by non-covalently linked oGTP in the factor and ribosome-dependent GTPase reaction, the factor was unable to utilize the covalently bound oGTP as a substrate. This indicates that the conformational flexibility in EF-2 required for the ribosomal activation of the GTPase was inhibited by the covalent attachment of the nucleotide to the factor. EF-2 cleaved at Arg66 were unable to form the high-affinity complex with ribosomes while retaining the ability to form the low-affinity complex and to hydrolyse GTP. The second cleavage at Lys571/Lys572 was accompanied by a total loss of both the low-affinity binding and the GTPase activity.     

Kinetic determination of the effects of ADP-ribosylation on the interaction of eukaryotic elongation factor 2 with ribosomes

The effect of ADP-ribosylation on the function of eukaryotic elongation factor 2 (EF-2) was investigated by kinetic analysis of the EF-2-catalyzed hydrolysis of GTP in the presence of ribosomes and by direct determination of the affinity of the modified factor for the ribosome. Under conditions where the concentration of EF-2 was rate-limiting, the ADP-ribosylation reduced the maximum rate of GTP hydrolysis and the second order rate constant Kcat/Km by approximately 50%. A similar decrease in Kcat and Kcat/Km was observed when the concentration of ribosomes were kept rate-limiting. The affinity of EF-2 for the pretranslocation type of ribosomes was reduced by 2 orders of magnitude after ADP-ribosylation. No effect was observed in the interaction with the post-translocation type of ribosomes, the ribosomal conformation responsible for activation of the EF-2-dependent GTPase. We conclude that the ADP-ribosylation affects both the association of the modified factor with pretranslocation ribosomes and the hydrolytic capacity of the factor.     

Biochemical analysis of a 5S rRNA-associated sub-particle from trypsinized eukaryotic ribosomes.

On limited trypsinization, eukaryotic ribosomes released sub-particles that comprised a 5S rRNA molecule and two peptides (a 32 kDa and a 14 kDa). By tryptic finger-printing and amino-terminal sequence analysis, these two peptides were determined to be derived from large subunit ribosomal protein L5 (rpL5). The 32 kDa peptide represents the rpL5 protein minus the amino terminal eight residues and the carboxyl terminal ends (approximately 21 residues), whereas the 14 kDa peptide comprised near the amino-terminal region. The time course of ribosome trypsinization revealed that the two peptides were released kinetically. The indicated that the amino and carboxyl terminal ends of rpL5 were the first to be hydrolyzed, suggesting that the two ends of the rpL5 protein were exposed on the surface of ribosomes. Exposure of the carboxyl-terminal end was confirmed by use of an anti-L5c antibody raised against the carboxyl terminal region of rpL5. The kinetic data also revealed that the nearby amino terminal region of rpL5 (represented by the 14 kDa peptide) was the last part of rpL5 to be hydrolyzed, which was considered to be the 5S rRNA binding site.     

Interface of the interaction of the middle domain of human translation termination factor eRF1 with eukaryotic ribosomes

Translation termination in eukaryotes is governed by the interaction of two, class 1 and class 2, polypeptide chain release factors with the ribosome. The middle (M) domain of the class 1 factor eRF1 contains the strictly conserved GGQ motif and is involved in hydrolysis of the peptidyl-tRNA ester bond in the peptidyl transferase center of the large ribosome subunit. Heteronuclear NMR spectroscopy was used to map the interaction interface of the M domain of human eRF1 with eukaryotic ribosomes. The protein was found to specifically interact with the 60S subunit, since no interaction was detected with the 40S subunit. The amino acid residues forming the interface mostly belong to long helix α1 of the M domain. Some residues adjacent to α1 and belonging to strand β5 and short helices α2 and α3 are also involved in the protein-ribosome contact. The functionally inactive G183A mutant interacted with the ribosome far more weakly as compared with the wild-type eRF1. The interaction interfaces of the two proteins were nonidentical. It was concluded that long helix α1 is functionally important and that the conformational flexibility of the GGQ loop is essential for the tight protein-ribosome contact.     

Biochemical and kinetic characterization of the RNA helicase activity of eukaryotic initiation factor 4A

Eukaryotic initiation factor (eIF) 4A is the prototypic member of the DEAD box family of proteins and has been proposed to act as an RNA helicase to unwind secondary structure in the 5'-untranslated region of eukaryotic mRNAs. Previous studies have shown that the RNA helicase activity of eIF4A is dependent on the presence of a second initiation factor, eIF4B. In this report, eIF4A has been demonstrated to function independently of eIF4B as an ATP-dependent RNA helicase. The biochemical and kinetic properties of this activity were examined. By using a family of RNA duplexes with an unstructured single-stranded region followed by a duplex region of increasing length and stability, it was observed that the initial rate of duplex unwinding decreased with increasing stability of the duplex. Furthermore, the maximum amount of duplex unwound also decreased with increasing stability. Results suggest that eIF4A acts in a non-processive manner. eIF4B and eIF4H were shown to stimulate the helicase activity of eIF4A, allowing eIF4A to unwind longer, more stable duplexes with both an increase in initial rate and maximum amount of duplex unwound. A simple kinetic model is proposed to explain the mechanism by which eIF4A unwinds RNA duplex structures in an ATP-dependent manner.     

Chemical, biochemical and genetic endeavours characterizing the interaction of sparsomycin with the ribosome.

Sparsomycin interaction with the ribosome and characteristics of the drug binding site in the particle were studied using chemical modification of the drug, affinity labeling methods and isolation of drug resistant mutants. The structure-function relationship studies, performed with a large number of drug derivatives, indicate that the drug interacts with the ribosome by its western and eastern moieties. The uracil ring, in the western end of the drug molecule, probably forms hydrogen bonds with the rRNA, while the apolar CH3-S-CH3 group in the eastern end interacts with a hydrophobic ribosomal domain that affinity labeling results seem to indicate is formed by protein. An increase in lipophilicity in this part of the antibiotic results in a dramatic increase in the inhibitory activity of the drug. The sparsomycin binding site is not accessible in free ribosomes, but the presence of an N-blocked amino acyl-tRNA at the P-site turns the particles capable of reversible interaction with the drug. After failure using Escherichia coli, a sparsomycin-resistant mutant was obtained by direct mutagenesis on Halobacterium halobium, a species with a unique copy of rRNA genes, stressing the role of rRNA on the drug interaction site.     

Evolution of prokaryotic and eukaryotic virulence effectors

Coevolutionary interactions between plants and their bacterial and eukaryotic pathogens are mediated by virulence effectors. These effectors face the daunting challenge of carrying out virulence functions, while also potentially exposing the pathogen to host defense systems. Very strong selective pressures are imposed by these competing roles, and the subsequent genetic changes leave their footprints in the extant allelic variation. This review examines the evolutionary processes that drive pathogen-host interactions as revealed by the genetic signatures left in virulence effectors, and speculate on the different pressures imposed on bacterial versus eukaryotic pathogens. We find numerous instances of positive selection for new allelic forms, and diversifying selection for genetic variability, which results in altered host-pathogen interactions. We also describe how the genetic structure of both bacterial and eukaryotic virulence effectors may contribute to their rapid generation and turnover.     

Immunochemical analysis of the structure of eukaryotic ribosomes

Summary Antibodies were prepared in rabbits and sheep to rat liver ribosomes, ribosomal subunits, and to mixtures of proteins from the particles. The antisera were characterized by quantitative immunoprecipitation, by passive hemagglutination, by immunodiffusion on Ouchterlony plates, and by immunoelectrophoresis. While all the antisera contained antibodies specific for ribosomal proteins, none had precipitating antibodies against ribosomal RNA. Rat liver ribosomal proteins were more immunogenic in sheep than rabbits, and the large ribosomal subunit and its proteins were more immunogenic than those of the 40S subparticle. Antisera specific for one or the other ribosomal subunit could be prepared; thus it is unlikely that there are antigenic determinants common to the proteins of the two subunits. When ribosomes, ribosomal subunits, or mixtures of proteins were used as antigens the sera contained antibodies directed against a large number of the ribosomal proteins.Abbreviations TP total proteins—used to designate mixtures of proteins from ribosomal particles, hence TP80 is a mixtures of all the proteins from 80S ribosomes - TP60 the proteins from 60S subunits - TP40 the proteins from 40S particles