Thiostrepton inhibits stable 70S ribosome binding and ribosome-dependent GTPase activation of elongation factor G and elongation factor 4

Thiostrepton, a macrocyclic thiopeptide antibiotic, inhibits prokaryotic translation by interfering with the function of elongation factor G (EF-G). Here, we have used 70S ribosome binding and GTP hydrolysis assays to study the effects of thiostrepton on EF-G and a newly described translation factor, elongation factor 4 (EF4). In the presence of thiostrepton, ribosome-dependent GTP hydrolysis is inhibited for both EF-G and EF4, with IC(50) values equivalent to the 70S ribosome concentration (0.15 μM). Further studies indicate the mode of thiostrepton inhibition is to abrogate the stable binding of EF-G and EF4 to the 70S ribosome. In support of this model, an EF-G truncation variant that does not possess domains IV and V was shown to possess ribosome-dependent GTP hydrolysis activity that was not affected by the presence of thiostrepton (>100 μM). Lastly, chemical footprinting was employed to examine the nature of ribosome interaction and tRNA movements associated with EF4. In the presence of non-hydrolyzable GTP, EF4 showed chemical protections similar to EF-G and stabilized a ratcheted state of the 70S ribosome. These data support the model that thiostrepton inhibits stable GTPase binding to 70S ribosomal complexes, and a model for the first step of EF4-catalyzed reverse-translocation is presented.     

G-418, an elongation inhibitor of 80 S ribosomes

The mode of action of the aminoglycoside G-418 was studied in wheat-germ, cell-free translation systems programmed with rat-liver polyadenylated RNA. Incorporation of amino acids into protein was effectively inhibited by G-418 in the microM concentration range. The inhibition pattern obtained was not uniform. The synthesis of polypeptides with higher molecular weights was more inhibited than that of smaller polypeptides. An identical inhibition pattern within a similar range of concentrations was obtained with cycloheximide, a known elongation inhibitor. Translation activity was abolished when the wheat-germ 80 S ribosomes were removed and could be partially reconstructed upon addition of the ribosomes. Incubation with G-418 prior to isolation yielded ribosomes defective in their reconstruction ability. The inhibition pattern was not uniform and exhibited again the same relationship between the size of a polypeptide and the extent of inhibition of its synthesis. Therefore, we suggest that in wheat-germ, cell-free translation systems G-418 affects the 80 S ribosomes and inhibits the elongation cycle.     

Role of elongation and secondary pathways in S6 amyloid fibril growth

The concerted action of a large number of individual molecular level events in the formation and growth of fibrillar protein structures creates a significant challenge for differentiating between the relative contributions of different self-assembly steps to the overall kinetics of this process. The characterization of the individual steps is, however, an important requirement for achieving a quantitative understanding of this general phenomenon which underlies many crucial functional and pathological pathways in living systems. In this study, we have applied a kinetic modeling approach to interpret experimental data obtained for the aggregation of a selection of site-directed mutants of the protein S6 from Thermus thermophilus. By studying a range of concentrations of both the seed structures, used to initiate the reaction, and of the soluble monomer, which is consumed during the growth reaction, we are able to separate unambiguously secondary pathways from primary nucleation and fibril elongation. In particular, our results show that the characteristic autocatalytic nature of the growth process originates from secondary processes rather than primary nucleation events, and enables us to derive a scaling law which relates the initial seed concentration to the onset of the growth phase.     

Ribosome-bound eukaryotic elongation factor 2 protects 5 S rRNA from modification.

The interaction between eukaryotic elongation factor eEF-2 and reconstituted 80 S ribosomes was investigated by analyzing the accessibility of 5 S ribosomal RNA for chemical and enzymatic modification. Ribosomes reconstituted from derived subunits were modified, and the positions of the modified sites were identified by primer extension using a 5 S rRNA-specific probe. All reactive sites were located between nucleotides 38 and 99, and most of them were found in putative single-stranded regions of the 5 S rRNA. Conversion of the ribosomes to the post-translocation type of particles by treatment with the translational inhibitor ricin resulted in the exposure of 3 additional bases for chemical modification, suggesting that the 5 S rRNA was more exposed in this type of ribosome. After binding of eEF-2 in complex with the non-hydrolyzable GTP analogue guanosine 5'-(beta, gamma-methylene)-triphosphate, most of the exposed bases in the 5 S rRNA were protected against both chemical and enzymatic modification.     

Brownian dynamics study of the association between the 70S ribosome and elongation factor G

Protein synthesis on the ribosome involves a number of external protein factors that bind at its functional sites. One key factor is the elongation factor G (EF-G) that facilitates the translocation of transfer RNAs between their binding sites, as well as advancement of the messenger RNA by one codon. The details of the EF-G/ribosome diffusional encounter and EF-G association pathway still remain unanswered. Here, we applied Brownian dynamics methodology to study bimolecular association in the bacterial EF-G/70S ribosome system. We estimated the EF-G association rate constants at 150 and 300 mM monovalent ionic strengths and obtained reasonable agreement with kinetic experiments. We have also elucidated the details of EF-G/ribosome association paths and found that positioning of the L11 protein of the large ribosomal subunit is likely crucial for EF-G entry to its binding site.     

Chemical crosslinking of elongation factor G to the 23S RNA in 70S ribosomes from Escherichia coli

Elongation factor G was crosslinked to the 23S RNA of 70S Escherichia coli ribosomes with the bifunctional, cleavable reagent diepoxybutane (DEB). The EF-G-23S RNA complex was isolated and digested with ribonuclease A. After digestion, an RNA fragment, protected by EF-G was cleaved from the complex and isolated. The nucleotide sequence of this RNA fragment was determined by partial ribonuclease digestion. It proved to be 27 nucleotides long and it could be identified with residues 1055 to 1081 of the nucleotide sequence of E. coli 23S RNA. In the presence of thiostrepton, which prevents binding of EF-G to the ribosome, there was a dramatic decrease in the yield of this complex.     

Cytological evidence for DNA chain elongation after UV irradiation in the S phase

Human cells irradiated with UV light synthesize lower molecular weight DNA than unirradiated cells. This reduction in molecular weight is greater in xeroderma pigmentosum (XP) cells than in normal cells. The molecular weight of DNA is further reduced by the addition of caffeine to XP cells. By several hours after irradiation, DNA fragments are barely detectable. Cells from excision-proficient and excision-deficient XP patients were studied autoradiographically to produce cytological evidence of DNA chain elongation. Replicate cultures with and without caffeine were synchronized and irradiated with UV light during the S phase. Caffeine was removed in G₂, and the cells were labeled with ³H-thymidine. Results showed significantly increased labeling during G₂ of excision-deficient XP cells. Labeling was dependent on both time of irradiation and presence of caffeine. The XP variant cells had no increase in labeling for any irradiation time.Published with the approval of the Director of the West Virginia Agricultural Experiment Station as Scientific Paper No. 1608. Supported by N.I.C. Grant TO1CA05170-10.     

Organization of genes for ribosomal proteins S7 and S12, elongation factors EF-Tu and EF-G in the cyanobacterium Spirulina platensis

The gene encoding ribosomal proteins S12 and probably S7 as well as protein synthesis elongation factors Tu (EF-Tu) and G (EF-G) of Spirulina platensis have been identified and cloned. Gene expression was determined for ribosomal protein S12 by genetic complementation of the appropriate Escherichia coli mutant, whereas for the EF-Tu gene it was determined by production of the protein in E. coli minicells. On the basis of these experiments we suggest the following gene order in the S. platensis chromosome: S12, S7, EF-G, EF-Tu.     

Genes for the ribosomal proteins S12 and S7 and elongation factors EF-G and EF-Tu of the cyanobacterium, Anacystis nidulans: structural homology between 16S rRNA and S7 mRNA

Summary A 6.5 kb region from the genome of the cyanobacterium, Anacystis nidulans 6301 was cloned using the tobacco chloroplast gene for ribosomal protein S12 as a probe. Sequence analysis revealed the presence of genes for ribosomal proteins S12 and S6 and elongation factors EF-G and EF-Tu in this DNA region. The arrangement is rps12 (124 codons)-167 bp spacer-rps7 (156 codons)-77 bp spacer-fus (694 codons)-26 bp spacer-tufA (409 codons), which is similar to that of the Escherichia coli str operon. The deduced amino acid sequences of the A. nidulans S12 and EF-Tu show high homology (72%–82%) with the E. coli and chloroplast counterparts while those of the A. nidulans S7 and EF-G give low homology (51%–59%). Striking structural homology was found between the potential S7 binding region of 16S rRNA and the beginning of S7 mRNA, suggesting that feedback regulation of rps7 expression operates in A. nidulans.     

Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation

An 11.7-A-resolution cryo-EM map of the yeast 80S.eEF2 complex in the presence of the antibiotic sordarin was interpreted in molecular terms, revealing large conformational changes within eEF2 and the 80S ribosome, including a rearrangement of the functionally important ribosomal intersubunit bridges. Sordarin positions domain III of eEF2 so that it can interact with the sarcin-ricin loop of 25S rRNA and protein rpS23 (S12p). This particular conformation explains the inhibitory action of sordarin and suggests that eEF2 is stalled on the 80S ribosome in a conformation that has similarities with the GTPase activation state. A ratchet-like subunit rearrangement (RSR) occurs in the 80S.eEF2.sordarin complex that, in contrast to Escherichia coli 70S ribosomes, is also present in vacant 80S ribosomes. A model is suggested, according to which the RSR is part of a mechanism for moving the tRNAs during the translocation reaction.     

Protein S3 fragments neighboring mRNA during elongation and termination of translation on the human ribosome

Protein S3 fragments were determined that crosslink to modified mRNA analogues in positions +5 to +12 relative to the first nucleotide in the P-site bound codon in model complexes mimicking states of ribosomes at the elongation and translation termination steps. The mRNA analogues contained a Phe codon UUU/UUC at the 5′-termini that could predetermine the position of the tRNA^Phe on the ribosome by the P-site binding and perfluorophenylazidobenzoyl group at a nucleotide in various positions 3′ of the UUU/UUC codon. The crosslinked S3 protein was isolated from 80S ribosomal complexes irradiated with mild UV light and subjected to cyanogen bromide—induced cleavage at methionine residues with subsequent identification of the crosslinked oligopeptides. An analysis of the positions of modified oligopeptides resulting from the cleavage showed that, in dependence on the positions of modified nucleotides in the mRNA analogue, the crosslinking sites were found in the N-terminal half of the protein (fragment 2–217) and/or in the C-terminal fragment 190–236; the latter reflects a new peculiarity in the structure of the mRNA binding center in the ribosome, unknown to date. The results of crosslinking did not depend on the type of A-site codon or on the presence of translation termination factor eRF1.     

A signal relay between ribosomal protein S12 and elongation factor EF-Tu during decoding of mRNA

Codon recognition by aminoacyl-tRNA on the ribosome triggers a process leading to GTP hydrolysis by elongation factor Tu (EF-Tu) and release of aminoacyl-tRNA into the A site of the ribosome. The nature of this signal is largely unknown. Here, we present genetic evidence that a specific set of direct interactions between ribosomal protein S12 and aminoacyl-tRNA, together with contacts between S12 and 16S rRNA, provide a pathway for the signaling of codon recognition to EF-Tu. Three novel amino acid substitutions, H76R, R37C, and K53E in Thermus thermophilus ribosomal protein S12, confer resistance to streptomycin. The streptomycin-resistance phenotypes of H76R, R37C, and K53E are all abolished by the mutation A375T in EF-Tu. A375T confers resistance to kirromycin, an antibiotic freezing EF-Tu in a GTPase activated state. H76 contacts aminoacyl-tRNA in ternary complex with EF-Tu and GTP, while R37 and K53 are involved in the conformational transition of the 30S subunit occurring upon codon recognition. We propose that codon recognition and domain closure of the 30S subunit are signaled through aminoacyl-tRNA to EF-Tu via these S12 residues.     

Stabilization by the 30S ribosomal subunit of the interaction of 50S subunits with elongation factor G and guanine nucleotide.

The role of the 30S ribosomal subunit in the formation of the complex ribosome-guanine nucleotide-elongation factor G (EF-G) has been examined in a great variety of experimental conditions. Our results show that at a large molar excess of EF-G or high concentrations of GTP or GDP, 50S ribosomal subunits are as active alone as with 30S subunits in the formation of the complex, while at lower concentrations of nucleotide or lower amounts of EF-G, addition of the 30S subunit stimulates greatly the reaction. The presence of the 30S ribosomal subunit can also moderate the inhibition of the 50S subunit activity that occurs by increasing moderately the concentrations of K+ and NH4+, and extends upward the concentration range of these monovalent cations in which complex formation is at maximum. The Mg2+ requirement for complex formation with the 50S subunit appears to be slightly less than that needed for association of the 30S and 50S ribosomal subunits. Measurement of the reaction rate constants of the complex formation shows that the 30S ribosomal subunit has only little effect on the initial association of EF-G and guanine nucleotide with the 50S subunit; but once this complex is formed, the 30S subunit increases its stability from 10- to 18-fold. It is concluded that stabilization of the interaction between EF-G and ribosome is a major function of the 30S subunit in the ribosome-EF-G GTPase reaction.     

Brassinolide-induced elongation and auxin

Segments from the hook and subhook zone of the stem of 6-day-old etiolated Pisum sativum L. cv. Victory Freezer seedlings were used to study the relationship between brassinolide and auxin in the promotion of elongation. Minor changes in exogenous indole-3-acetic acid or4-chloroindole-3-acetic acid concentration affected the kinetics markedly and the ethylene generator ethephon overcame brassinolide-induced elongation in an antagonistic interaction. Brassinolide-induced elongation was markedly inhibited by low concentrations of the cellulose biosynthesis inhibitor 2,6-dichlorobenzonitrile, and diagnostic concentrations of the antiauxin 2-( p -chlorophenoxy) isobutyric acid did not affect brassinolide-induced elongation. As the characteristics of auxin-induced growth are not displayed in brassinolide-induced elongation of the upper stem segment, it is proposed that brassinolide does not depend on auxin as a mediator in the promotion of elongation of younger tissues but that it can interact in a very complex manner with auxin. In the elongation of more mature tissues, and in bending responses, brassinolide probably accelerates auxin effects. When split, the upper stem segment was unusual in its lack of specific response to growth regulators, and the slight relief of epidermal tension.     

Identification of genes for elongation factor Ts and ribosomal protein S2 in E. coli

The structural gene for elongation factor EF-TS (tsf) and that for ribosomal protein S2 (rpsB) have been identified in E. coli. Both genes are carried by λ transducing phages that have been isolated as dapD^?polC⁺ transducing phages. Synthesis of both S2 and EF-Ts was demonstrated in ultraviolet light-irradiated E. coli cells infected with these phages. Experiments were also done using other transducing phages that carry dapD⁺ but not polC⁺. The data indicate that both the tsf and rpsB genes map near dapD at about 4 min on the E. coli genetic map. This location is different from the two chromosomal locations, the str-spc region and the rif region, where many ribosomal protein genes, the genes for RNA polymerase components, as well as other elongation factor genes (fus, tufA, and tufB) are located.     

Eukaryote-specific motif of ribosomal protein S15 neighbors A site codon during elongation and termination of translation

The eukaryotic ribosomal protein S15 is a key component of the decoding site in contrast to its prokaryotic counterpart, S19p, which is located away from the mRNA binding track on the ribosome. Here, we determined the oligopeptide of S15 neighboring the A site mRNA codon on the human 80S ribosome with the use of mRNA analogues bearing perfluorophenyl azide-modified nucleotides in the sense or stop codon targeted to the 80S ribosomal A site. The protein was cross-linked to mRNA analogues in specific ribosomal complexes that were obtained in the presence of eRF1 in the experiments with mRNAs bearing stop codon. Digestion of modified S15 with various specific proteolytic agents followed by identification of the resulting modified oligopeptides showed that cross-link was in C-terminal fragment in positions 131–145, most probably, in decapeptide 131-PGIGATHSSR-140. The position of cross-linking site on the S15 protein did not depend on the nature of the A site-bound codon (sense or stop codon) and on the presence of polypeptide chain release factor eRF1 in the ribosomal complexes with mRNA analogues bearing a stop codon. The results indicate an involvement of the mentioned decapeptide in the formation of the ribosomal decoding site during elongation and termination of translation. Alignment of amino acid sequences of eukaryotic S15 and its prokaryotic counterpart, S19p from eubacteria and archaea, revealed that decapeptide PGIGATHSSR in positions 131–140 is strongly conserved in eukaryotes and has minor variations in archaea but has no homology with any sequence in C-terminal part of eubacterial S19p, which suggests involvement of the decapeptide in the translation process in a eukaryote-specific manner.