guanosine-triphosphate and mocimycin

Elfamycins are a relatively understudied group of antibiotics that target the essential process of translation through impairment of EF-Tu function. For the most part, the utility of these compounds has been as laboratory tools for the study of EF-Tu and the ribosome, as their poor pharmacokinetic profile and solubility has prevented implementation as therapeutic agents. However, due to the slowing of the antibiotic pipeline and the rapid emergence of resistance to approved antibiotics, this group is being reconsidered. Some researchers are using screens for novel naturally produced variants, while others are making directed, systematic chemical improvements on publically disclosed compounds. As an example of the latter approach, a GE2270 A derivative, LFF571, has completed phase 2 clinical trials, thus demonstrating the potential for elfamycins to become more prominent antibiotics in the future.

Topics: Actinomycetales; Actinomycetales Infections; Aminoglycosides; Anti-Bacterial Agents; Drug Design; Escherichia coli; Guanosine Triphosphate; Peptide Elongation Factor Tu; Peptides, Cyclic; Polyenes; Pyridones; Ribosomes; Thiazoles

In bacteria, ribosomes stalled on truncated mRNAs are rescued by transfer-messenger RNA (tmRNA) and its protein partner SmpB. Acting like tRNA, the aminoacyl-tmRNA/SmpB complex is delivered to the ribosomal A site by EF-Tu and accepts the transfer of the nascent polypeptide. Although SmpB binding within the decoding center is clearly critical for licensing tmRNA entry into the ribosome, it is not known how activation of EF-Tu occurs in the absence of a codon-anticodon interaction. A recent crystal structure revealed that SmpB residue His136 stacks on 16S rRNA nucleotide G530, a critical player in the canonical decoding mechanism. Here we use pre-steady-state kinetic methods to probe the role of this interaction in ribosome rescue. We find that although mutation of His136 does not reduce SmpB's affinity for the ribosomal A-site, it dramatically reduces the rate of GTP hydrolysis by EF-Tu. Surprisingly, the same mutation has little effect on the apparent rate of peptide-bond formation, suggesting that release of EF-Tu from the tmRNA/SmpB complex on the ribosome may occur prior to GTP hydrolysis. Consistent with this idea, we find that peptidyl transfer to tmRNA is relatively insensitive to the antibiotic kirromycin. Taken together, our studies provide a model for the initial stages of ribosomal rescue by tmRNA.

Topics: Amino Acid Sequence; Amino Acid Substitution; Anti-Bacterial Agents; Base Sequence; Escherichia coli; Gene Expression Regulation, Bacterial; Guanosine Triphosphate; Hydrolysis; Kinetics; Mutagenesis, Site-Directed; Peptide Elongation Factor Tu; Point Mutation; Protein Binding; Pyridones; Ribosomes; RNA-Binding Proteins; RNA, Bacterial

During protein synthesis, translation elongation factor Tu (Ef-Tu) is responsible for the selection and binding of the cognate aminoacyl-tRNA to the acceptor site on the ribosome. The activity of Ef-Tu is dependent on its interaction with GTP. Posttranslational modifications, such as phosphorylation, are known to regulate the activity of Ef-Tu in several prokaryotes. Although a study of the Mycobacterium tuberculosis phosphoproteome showed Ef-Tu to be phosphorylated, the role of phosphorylation in the regulation of Ef-Tu has not been studied. In this report, we show that phosphorylation of M. tuberculosis Ef-Tu (MtbEf-Tu) by PknB reduced its interaction with GTP, suggesting a concomitant reduction in the level of protein synthesis. Overexpression of PknB in Mycobacterium smegmatis indeed reduced the level of protein synthesis. MtbEf-Tu was found to be phosphorylated by PknB on multiple sites, including Thr118, which is required for optimal activity of the protein. We found that kirromycin, an Ef-Tu-specific antibiotic, had a significant effect on the nucleotide binding of unphosphorylated MtbEf-Tu but not on the phosphorylated protein. Our results show that the modulation of the MtbEf-Tu-GTP interaction by phosphorylation can have an impact on cellular protein synthesis and growth. These results also suggest that phosphorylation can change the sensitivity of the protein to the specific inhibitors. Thus, the efficacy of an inhibitor can also depend on the posttranslational modification(s) of the target and should be considered during the development of the molecule.

Topics: Electrophoresis, Gel, Two-Dimensional; Guanosine Triphosphate; Immunoblotting; Mycobacterium tuberculosis; Peptide Elongation Factor Tu; Phosphoproteins; Phosphorylation; Phosphothreonine; Protein Binding; Protein Serine-Threonine Kinases; Pyridones

Elongation factor (EF-) Tu.GTP is the carrier of aminoacyl-tRNA to the programmed ribosome. Enacyloxin IIa inhibits bacterial protein synthesis by hindering the release of EF-Tu.GDP from the ribosome. The crystal structure of the Escherichia coli EF-Tu.guanylyl iminodiphosphate (GDPNP).enacyloxin IIa complex at 2.3 A resolution presented here reveals the location of the antibiotic at the interface of domains 1 and 3. The binding site overlaps that of kirromycin, an antibiotic with a structure that is unrelated to enacyloxin IIa but that also inhibits EF-Tu.GDP release. As one of the major differences, the enacyloxin IIa tail borders a hydrophobic pocket that is occupied by the longer tail of kirromycin, explaining the higher binding affinity of the latter. EF-Tu.GDPNP.enacyloxin IIa shows a disordered effector region that in the Phe-tRNAPhe.EF-Tu (Thermus aquaticus).GDPNP.enacyloxin IIa complex, solved at 3.1 A resolution, is stabilized by the interaction with tRNA. This work clarifies the structural background of the action of enacyloxin IIa and compares its properties with those of kirromycin, opening new perspectives for structure-guided design of novel antibiotics.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Escherichia coli Proteins; Guanosine Triphosphate; Peptide Elongation Factor Tu; Polyenes; Pyridones; RNA, Transfer; Thermus

The antibiotic pulvomycin is an inhibitor of protein synthesis that prevents the formation of the ternary complex between elongation factor (EF-) Tu.GTP and aminoacyl-tRNA. In this report, novel aspects of its action on EF-Tu are described. Pulvomycin markedly affects the equilibrium and kinetics of the EF-Tu-nucleotide interaction, particularly of the EF-Tu.GTP complex. The binding affinity of EF-Tu for GTP is increased 1000 times, mainly as the consequence of a dramatic decrease in the dissociation rate of this complex. In contrast, the affinity for GDP is decreased 10-fold due to a marked increase in the dissociation rate of EF-Tu.GDP (25-fold) that mimics the action of EF-Ts, the GDP/GTP exchange factor of EF-Tu. The effects of pulvomycin and EF-Ts can coexist and are simply additive, supporting the conclusion that these two ligands interact with different sites of EF-Tu. This is further confirmed on native PAGE by the ability of EF-Tu to bind the EF-Ts and the antibiotic simultaneously. Pulvomycin enhances the intrinsic EF-Tu GTPase activity, like kirromycin, though to a much more modest extent. As with kirromycin, this stimulation depends on the concentration and nature of the monovalent cations, Li(+) being the most effective one, followed by Na(+), K(+), and NH(4)(+). In the presence of pulvomycin (in contrast to kirromycin), aa-tRNA and/or ribosomes do not enhance the GTPase activity of EF-Tu. The property of pulvomycin to modify selectively the conformation(s) of EF-Tu is also supported by its effect on heat- and urea-dependent denaturation, and tryptic digestion of the protein. Specific differences and similarities between the action of pulvomycin and the other EF-Tu-specific antibiotics are described and discussed.

Topics: Aminoglycosides; Anti-Bacterial Agents; Binding Sites; Enzyme Inhibitors; Enzyme Stability; GTP Phosphohydrolases; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Peptide Elongation Factor Tu; Peptide Elongation Factors; Peptides, Cyclic; Protein Denaturation; Pyridones; Thiazoles; Trypsin; Urea

Bacterial ribosomes stalled on defective messenger RNAs (mRNAs) are rescued by tmRNA, an approximately 300-nucleotide-long molecule that functions as both transfer RNA (tRNA) and mRNA. Translation then switches from the defective message to a short open reading frame on tmRNA that tags the defective nascent peptide chain for degradation. However, the mechanism by which tmRNA can enter and move through the ribosome is unknown. We present a cryo-electron microscopy study at approximately 13 to 15 angstroms of the entry of tmRNA into the ribosome. The structure reveals how tmRNA could move through the ribosome despite its complicated topology and also suggests roles for proteins S1 and SmpB in the function of tmRNA.

Topics: Alanine; Anticodon; Bacterial Proteins; Codon; Codon, Terminator; Cryoelectron Microscopy; Guanosine Triphosphate; Image Processing, Computer-Assisted; Models, Molecular; Nucleic Acid Conformation; Open Reading Frames; Peptide Elongation Factor Tu; Protein Biosynthesis; Pyridones; Ribosomal Proteins; Ribosomes; RNA-Binding Proteins; RNA, Bacterial; RNA, Messenger; RNA, Transfer; Thermus thermophilus

The G13A substitution in the G13XXXXGK[T,S] consensus sequence of the elongation factor 1 alpha from the archaeon Sulfolobus solfataricus (SsEF-1 alpha) was introduced in order to study the reasons for selective differences found in the homologous consensus element AXXXXGK[T,S] of the other elongation factor EF-2 or EF-G. In a previous work, it was shown that the main effect of the A26G mutation was the activation of the intrinsic GTPase of SsEF-2 [De Vendittis, E., Adinolfi, B. S., Amatruda, M. R., Raimo, G., Masullo, M., and Bocchini, V. (1994) Eur. J. Biochem. 262, 600-605]. In this work, we found that, compared to the wild-type factor (SsEF-1 alpha wt), G13ASsEF-1 alpha shows (i) a reduced rate of [(3)H]Phe polymerization that was probably due to its reduced ability to form a ternary complex with heterologous aa-tRNA and (ii) a reduced intrinsic GTPase activity that was stimulated by high concentrations of NaCl (GTPase(Na)) [Masullo, M., De Vendittis, E., and Bocchini, V. (1994) J. Biol. Chem. 269, 20376-20379]. In addition, G13ASsEF-1 alpha showed an increased affinity for GDP and GTP. Surprisingly, the decreased intrinsic GTPase(Na) of G13ASsEF-1 alpha can be partially restored by kirromycin, an effect not found for SsEF-1 alpha wt. The temperature inducing a 50% denaturation of G13ASsEF-1 alpha was somewhat lower (-5 degrees C) than that of SsEF-1 alpha wt, and the decrease in its thermophilicity was slightly more accentuated (-10 degrees C). These results indicate that the nature of the residue in position 13 is important for the functional and physical properties of SsEF-1 alpha.

Topics: Anti-Bacterial Agents; GTP Phosphohydrolases; Guanine; Guanosine Diphosphate; Guanosine Triphosphate; Hot Temperature; Hydrolysis; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Peptide Elongation Factor 1; Plasmids; Protein Binding; Pyridones; Sulfolobus; Temperature; Time Factors

During the elongation cycle of protein biosynthesis, the specific amino acid coded for by the mRNA is delivered by a complex that is comprised of the cognate aminoacyl-tRNA, elongation factor Tu and GTP. As this ternary complex binds to the ribosome, the anticodon end of the tRNA reaches the decoding center in the 30S subunit. Here we present the cryo- electron microscopy (EM) study of an Escherichia coli 70S ribosome-bound ternary complex stalled with an antibiotic, kirromycin. In the cryo-EM map the anticodon arm of the tRNA presents a new conformation that appears to facilitate the initial codon-anticodon interaction. Furthermore, the elbow region of the tRNA is seen to contact the GTPase-associated center on the 50S subunit of the ribosome, suggesting an active role of the tRNA in the transmission of the signal prompting the GTP hydrolysis upon codon recognition.

Topics: Anticodon; Codon; Cryoelectron Microscopy; Escherichia coli; Escherichia coli Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Image Processing, Computer-Assisted; Macromolecular Substances; Models, Molecular; Nucleic Acid Conformation; Peptide Chain Elongation, Translational; Peptide Elongation Factor Tu; Protein Conformation; Pyridones; Ribosomes; RNA, Transfer; RNA, Transfer, Amino Acyl; RNA, Transfer, Phe; Structure-Activity Relationship

The influence of divalent metal ions on the intrinsic and kirromycin-stimulated GTPase activity in the absence of programmed ribosomes and on nucleotide binding affinity of elongation factor Tu (EF-Tu) from Thermus thermophilus prepared as the nucleotide- and Mg(2+)-free protein has been investigated. The intrinsic GTPase activity under single turnover conditions varied according to the series: Mn(2+) (0.069 min(-1)) > Mg(2+) (0.037 min(-1)) approximately no Me(2+) (0.034 min(-1)) > VO(2+) (0.014 min(-1)). The kirromycin-stimulated activity showed a parallel variation. Under multiple turnover conditions (GTP/EF-Tu ratio of 10:1), Mg(2+) retarded the rate of hydrolysis in comparison to that in the absence of divalent metal ions, an effect ascribed to kinetics of nucleotide exchange. In the absence of added divalent metal ions, GDP and GTP were bound with equal affinity (K(d) approximately 10(-7) m). In the presence of added divalent metal ions, GDP affinity increased by up to two orders of magnitude according to the series: no Me(2+) < VO(2+) < Mn(2+) approximately Mg(2+) whereas the binding affinity of GTP increased by one order of magnitude: no Me(2+) < Mg(2+) < VO(2+) < Mn(2+). Estimates of equilibrium (dissociation) binding constants for GDP and GTP by EF-Tu on the basis of Scatchard plot analysis, together with thermodynamic data for hydrolysis of triphosphate nucleotides (Phillips, R. C., George, P., and Rutman, R. J. (1969) J. Biol. Chem. 244, 3330-3342), showed that divalent metal ions stabilize the EF-Tu.Me(2+).GDP complex over the protein-free Me(2+).GDP complex in solution, with the effect greatest in the presence of Mg(2+) by approximately 10 kJ/mol. These combined results show that Mg(2+) is not a catalytically obligatory cofactor in intrinsic and kirromycin-stimulated GTPase action of EF-Tu in the absence of programmed ribosomes, which highlights the differential role of Mg(2+) in EF-Tu function.

Topics: Anti-Bacterial Agents; Catalysis; Dose-Response Relationship, Drug; GTP Phosphohydrolases; Guanosine Triphosphate; Ions; Kinetics; Magnesium; Models, Chemical; Nucleotides; Peptide Elongation Factor Tu; Protein Binding; Pyridones; Spectrum Analysis; Thermodynamics; Thermus thermophilus; Time Factors

Protein kinase associated with ribosomes of streptomycetes phosphorylates 11 ribosomal proteins. Phosphorylation activity of protein kinase reaches its maximum at the end of exponential phase of growth. When (32)P-labeled cells from the end of exponential phase of growth were transferred to a fresh medium, after 2 h of cultivation ribosomal proteins lost more than 90% of (32)P and rate of polypeptide synthesis increases twice. Protein kinase cross-reacting with antibody raised against protein kinase C was partially purified from 1 M NH(4)Cl wash of ribosomes and used to phosphorylation of ribosomes. Phosphorylation of 50S subunits (L2, L3, L7, L16, L21, L23, and L27) had no effect on the integrity of subunits but affects association with 30 to 70S monosomes. In vitro system derived from ribosomal subunits was used to examine the activity of phosphorylated 50S at poly(U) translation. Replacement unphosphorylated 50S with 50S possessed of phosphorylated r-proteins leads to the reduction of polypeptide synthesis of about 52%. The binding of N-Ac[(14)C]Phe-tRNA to A-site of phosphorylated ribosomes is not affected but the rate of peptidyl transferase is more than twice lower than that in unphosphorylated ribosomes. These results provide evidence that phosphorylation of ribosomal proteins is involved in mechanisms regulating the translational system of Streptomyces collinus.

Topics: Anti-Bacterial Agents; Binding Sites; Cell-Free System; Electrophoresis, Gel, Two-Dimensional; Guanosine Triphosphate; Peptidyl Transferases; Phosphorylation; Protein Binding; Protein Biosynthesis; Protein Kinases; Proteins; Pyridones; Ribosomes; RNA, Transfer, Amino Acyl; Signal Transduction; Streptomyces; Time Factors

A 4.5-kb BamHI fragment of chromosomal DNA of Streptomyces collinus containing gene ftsZ was cloned and sequenced. Upstream of ftsZ are localized genes ftsQ, murG, and ftsW, and downstream is yfiH. Gene ftsA is not adjacent to ftsZ or other genes of the cloned fragment. Protein FtsZ was isolated and characterized with respect to its binding to GTP and GTPase activity. The binding of GTP to FtsZ was Ca(2+) or Mg(2+) dependent with an optimum at 10 mM. The rate of GTP hydrolysis by FtsZ was stimulated by KCl. The presence of Ca(2+) (3-5 mM) resulted in a significant increase of GTPase activity. Higher concentrations of Ca(2+) than 5 mM had an inhibitory effect on GTPase activity. These results indicate that divalent ions (Ca(2+) or Mg(2+)) can be involved in regulation of GTP binding and hydrolysis of FtsZ. The maximum level of FtsZ was detected in aerial mycelium when spiral loops and sporulation septa were formed. FtsZ is degraded after finishing sporulation septa.

Topics: Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Bacterial Proteins; Cell Cycle; Cytoskeletal Proteins; DNA, Bacterial; Escherichia coli Proteins; Genes, Bacterial; GTP Phosphohydrolases; Guanosine Triphosphate; Membrane Proteins; Multigene Family; N-Acetylglucosaminyltransferases; Pyridones; Sequence Analysis; Streptomyces

Elongation factor (EF) Tu Thr-25 is a key residue binding the essential magnesium complexed to nucleotide. We have characterized mutations at this position to the related Ser and to Ala, which abolishes the bond to Mg2+, and a double mutation, H22Y/T25S. Nucleotide interaction was moderately destabilized in EF-Tu(T25S) but strongly in EF-Tu(T25A) and EF-Tu(H22Y/T25S). Binding Phe-tRNAPhe to poly(U).ribosome needed a higher magnesium concentration for the latter two mutants but was comparable at 10 mM MgCl2. Whereas EF-Tu(T25S) synthesized poly(Phe), as effectively as wild type, the rate was reduced to 50% for EF-Tu(H22Y/T25S) and was, surprisingly, still 10% for EF-Tu(T25A). In contrast, protection of Phe-tRNAPhe against spontaneous hydrolysis by the latter two mutants was very low. The intrinsic GTPase in EF-Tu(H22Y/T25S) and (T25A) was reduced, and the different responses to ribosomes and kirromycin suggest that stimulation by these two agents follows different mechanisms. Of the mutants, only EF-Tu(T25A) forms a more stable complex with EF-Ts than wild type. This implies that stabilization of the EF-Tu.EF-Ts complex is related to the inability to bind Mg2+, rather than to a decreased nucleotide affinity. These results are discussed in the light of the three-dimensional structure. They emphasize the importance of the Thr-25-Mg2+ bond, although its absence is compatible with protein synthesis and thus with an active overall conformation of EF-Tu.

Topics: GTP Phosphohydrolase-Linked Elongation Factors; Guanosine Diphosphate; Guanosine Triphosphate; Magnesium; Models, Molecular; Peptide Elongation Factor Tu; Protein Binding; Protein Conformation; Pyridones; Recombinant Fusion Proteins; Ribosomes; RNA, Transfer, Phe; Structure-Activity Relationship; Threonine

The properties of variants of elongation factor (EF) Tu mutated at three positions implicated in its GTPase activity are presented. Mutation I60A, which reduces one wing of a "hydrophobic barrier" screening off the nucleophilic water molecule found at the GTP gamma-phosphate, causes a reduction of the intrinsic GTPase activity contrary to prediction and has practically no influence on other properties. Mutation D80N, which in the isolated G-domain of EF-Tu caused a strong stimulation of the intrinsic GTPase, reduces this activity in the intact molecule. However, whereas for wild-type EF-Tu complex formation with aa-tRNA reduces the GTPase, EF-Tu[D80N] shows a strongly increased activity when bound to Phe-tRNA. Moreover, ribosomes or kirromycin can stimulate its GTPase up to the same level as for wild-type. This indicates that a local destabilization of the magnesium binding network does not per se cause an increased GTPase but does affect its tight regulation. Interestingly, mutant D80N sequestrates EF-Ts by formation of a more stable complex. Substitutions T61A and T61N induce low intrinsic GTPase, and the stimulation by ribosome is less for T61A than for T61N but still detectable, while kirromycin stimulates the GTPase of both mutants equally. This provides more evidence that stimulation by kirromycin and ribosomes follows a different mechanism. The functional implications of these mutations are discussed in the context of a transition state mechanism for catalysis. An alternative structural explanation for the strong conservation of Ile-60 is proposed.

Topics: Aspartic Acid; Binding Sites; Escherichia coli; Esters; GTP Phosphohydrolases; Guanosine Diphosphate; Guanosine Triphosphate; Isoleucine; Magnesium; Models, Molecular; Mutagenesis, Site-Directed; Peptide Elongation Factor Tu; Peptides; Pyridones; Recombinant Proteins; Ribosomes; RNA, Transfer, Phe; Threonine

For clarification of the action of a new antibiotic, the analysis of resistant mutants is often indispensable. For enacyloxin IIa we discovered four resistant elongation factor Tu (EF-Tu) species in Escherichia coli with the mutations Q124K, G316D, Q329H, and A375T, respectively. They revealed that enacyloxin IIa sensitivity is dominant in a mixed population of resistant and wild-type EF-Tus. This points to an inhibition mechanism in which EF-Tu is the dominant target of enacyloxin IIa and in which a ribosome with a sensitive EF-Tu blocks mRNA translation for upstream ribosomes with resistant EF-Tus, a mechanism similar to that of the unrelated antibiotic kirromycin. Remarkably, the same mutations are also linked to kirromycin resistance, though the order of their levels of resistance is different from that for enacyloxin IIa. Among the mutant EF-Tus, three different resistance mechanisms can be distinguished: (i) by obstructing enacyloxin IIa binding to EF-Tu. GTP; (ii) by enabling the release of enacyloxin IIa after GTP hydrolysis; and (iii) by reducing the affinity of EF-Tu.GDP. enacyloxin IIa for aminoacyl-tRNA at the ribosomal A-site, which then allows the release of EF-Tu.GDP.enacyloxin IIa. Ala375 seems to contribute directly to enacyloxin IIa binding at the domain 1-3 interface of EF-Tu.GTP, a location that would easily explain the pleiotropic effects of enacyloxin IIa on the functioning of EF-Tu.

Topics: Escherichia coli; GTP Phosphohydrolases; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Models, Molecular; Peptide Elongation Factor Tu; Phenylalanine; Polyenes; Polymers; Protein Biosynthesis; Pyridones; Ribosomes; RNA, Transfer, Amino Acyl; Structure-Activity Relationship

Elongation factor Tu (EF-Tu) from E. coli was coupled to activated CH Sepharose 4B. The immobilized EF-Tu maintained most of the guanosine nucleotide binding activity, but its ability to bind aminoacyl-tRNA depended on the type of complex used in the coupling reaction. The EF-Tu.GTP.aminoacyl-tRNA.kirromycin complex yielded an immobilized factor that was much more active in binding aminoacyl-tRNA than that obtained by coupling EF-Tu.GDP or EF-Tu.GDP.kirromycin to CH Sepharose 4B. Like the free factor, the immobilized EF-Tu.GTP did bind aminoacyl-tRNAs, but not unacylated tRNAs or N-acylated-aminoacyl-tRNAs. The antibiotic kirromycin was used to obtain the rapid conversion of EF-Tu.GDP to EF-Tu.GTP and the release of aminoacyl-tRNA from the matrix-bound EF-Tu by GDP. When total tRNA was aminoacylated by one amino acid only, a column of immobilized EF-Tu-GTP.kirromycin allowed the isolation of the aminoacylated tRNA from bulk tRNA. A rapid and nearly quantitative recovery of highly purified tRNA isoacceptors for various amino acids was obtained in one chromatographic step.

Topics: Chromatography, Affinity; Escherichia coli; Evaluation Studies as Topic; Guanosine Triphosphate; Ligands; Peptide Elongation Factor Tu; Pyridones; RNA, Transfer, Amino Acyl; Sepharose

The delivery of a specific amino acid to the translating ribosome is fundamental to protein synthesis. The binding of aminoacyl-transfer RNA to the ribosome is catalysed by the elongation factor Tu (EF-Tu). The elongation factor, the aminoacyl-tRNA and GTP form a stable 'ternary' complex that binds to the ribosome. We have used electron cryomicroscopy and angular reconstitution to visualize directly the kirromycin-stalled ternary complex in the A site of the 70S ribosome of Escherichia coli. Electron cryomicroscopy had previously given detailed ribosomal structures at 25 and 23 A resolution, and was used to determine the position of tRNAs on the ribosome. In particular, the structures of pre-translocational (tRNAs in A and P sites) and post-translocational ribosomes (P and E sites occupied) were both visualized at a resolution of approximately 20 A. Our three-dimensional reconstruction at 18 A resolution shows the ternary complex spanning the inter-subunit space with the acceptor domain of the tRNA reaching into the decoding centre. Domain 1 (the G domain) of the EF-Tu is bound both to the L7/L12 stalk and to the 50S body underneath the stalk, whereas domain 2 is oriented towards the S12 region on the 30S subunit.

Topics: Crystallography, X-Ray; Escherichia coli; Guanosine Triphosphate; Models, Molecular; Nucleic Acid Conformation; Peptide Elongation Factor Tu; Protein Conformation; Pyridones; Ribosomes; RNA, Transfer

We have mutated lysine 2 and arginine 7 in elongation factor Tu from Escherichia coli separately either to alanine or glutamic acid. The aim of the work was to reveal the possible interactions between the conserved N-terminal part of the molecule, which is rich in basic residues and aminoacyl-tRNA. The enzymatic characterization, comprising GDP and GTP temperature stability assays and measurement of nucleotide dissociation and association rate constants, GTPase activity and aminoacyl-tRNA binding, shows that position 2 is not involved in aminoacyl-tRNA binding, while position 7 is necessary to accomplish this activity. Furthermore, arginine 7 seems to play a role in regulating the binding of GTP. The three-dimensional structure of the ternary complex, EF-Tu:GTP:Phe-tRNAPhe, involving Thermus aquaticus EF-Tu and yeast Phe-tRNA(Phe), shows that Arg7 is in a position which permits salt bridge formation with Asp284, thus binding the N-terminus tightly to domain 2. We propose that this interaction is needed for aminoacyl-tRNA binding, and also for completing the structural rearrangement, which takes place when the factor switches from its GDP to its GTP form.

Topics: DNA Mutational Analysis; Escherichia coli; GTP Phosphohydrolase-Linked Elongation Factors; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Models, Molecular; Mutagenesis; Nucleic Acid Conformation; Peptide Elongation Factor Tu; Protein Conformation; Pyridones; RNA, Transfer, Phe

Kirromycin inhibits bacterial protein synthesis by acting on elongation factor Tu (EF-Tu). Complexes of the antibiotic, Phe-tRNA(Phe), the guanosine triphosphate analog GDPNP, and mesophilic (Escherichia coli), as well as thermophilic (Thermus thermophilus) EF-Tu were isolated. Crystallization was achieved at 4 degrees C, pH 6.4, using ammonium sulphate as precipitant. Crystallographic data were recorded at cryogenic temperature on crystals exposed to synchrotron radiation. Crystals of the thermophilic complex are based on a rhombohedral lattice with cell dimensions of 137.3 A, and angles of 54.0 degrees. Although related, these cell parameters are different from those found in the crystals of the recently solved structure of the ternary complex of Phe-tRNA(Phe), GDPNP, and Thermus aquaticus EF-Tu (Nissen, P., Kjeldgaard, M., Thirup, S., Polekhina, G., Reshetnikova, L., Clark, B.F. and Nyborg, J. (1995) Science 270, 1464-1472 [1]), possibly indicating some allosteric effect caused by kirromycin. Crystals of the mesophilic complex belong to the cubic space P432, with cell axis of 196.26 A. In both cases, the crystals contain one complex per asymmetric unit.

Topics: Guanosine Triphosphate; Peptide Elongation Factor Tu; Pyridones; RNA, Transfer, Amino Acyl; X-Ray Diffraction

The mechanisms by which elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA to the A site of the ribosome and, in particular, how GTP hydrolysis by EF-Tu is triggered on the ribosome, are not understood. We report steady-state and time-resolved fluorescence measurements, performed in the Escherichia coli system, in which the interaction of the complex EF-Tu.GTP.Phe-tRNAPhe with the ribosomal A site is monitored by the fluorescence changes of either mant-dGTP [3'-O-(N-methylanthraniloyl)-2-deoxyguanosine triphosphate], replacing GTP in the complex, or of wybutine in the anticodon loop of the tRNA. Additionally, GTP hydrolysis is measured by the quench-flow technique. We find that codon-anticodon interaction induces a rapid rearrangement within the G domain of EF-Tu around the bound nucleotide, which is followed by GTP hydrolysis at an approximately 1.5-fold lower rate. In the presence of kirromycin, the activated conformation of EF-Tu appears to be frozen. The steps following GTP hydrolysis--the switch of EF-Tu to the GDP-bound conformation, the release of aminoacyl-tRNA from EF-Tu to the A site, and the dissociation of EF-Tu-GDP from the ribosome--which are altogether suppressed by kirromycin, are not distinguished kinetically. The results suggest that codon recognition by the ternary complex on the ribosome initiates a series of structural rearrangements resulting in a conformational change of EF-Tu, possibly involving the effector region, which, in turn, triggers GTP hydrolysis.

Topics: Anticodon; Binding Sites; Codon; Escherichia coli; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Nucleic Acid Conformation; ortho-Aminobenzoates; Peptide Elongation Factor Tu; Protein Conformation; Pyridones; Ribosomes; RNA, Transfer, Amino Acyl

Pulvomycin is a strong inhibitor of protein synthesis, known to prevent the binding of aminoacyl-tRNA to elongation factor Tu.GTP (EF-Tu.GTP). Recently, three pulvomycin-resistant mutant strains have been isolated by targeted mutagenesis of the tufA gene resulting in EF-Tu substitutions at positions 230, 333 or 334. In order to analyze the functions of arginine residues located in domain II, with respect to pulvomycin resistance and the interaction with aminoacyl-tRNA, we have investigated the effect of the substitutions of the highly conserved residues Arg230 and Arg233 by site-directed mutagenesis. We have purified two mutants species, [R233S]EF-TuHis and [R230V, R233F]EF-TuHis, both with a C-terminal histidine extension to enable purification by Ni2+ affinity chromatography. In this study, we describe the in vitro characterization of these mutant proteins. The results show that the concomitant substitution of residues at positions 230 and 233, dramatically increases the pulvomycin resistance. Preliminary evidence is presented that protein synthesis is inhibited by an EF-Tu.GDP.pulvomycin complex rather than by EF-Tu.GTP.pulvomycin. Moreover, the mutant [R230V, R233F]EF-TuHis shows a stronger protection of the ester bond of aminoacyl-tRNA than wild-type EF-Tu.

Topics: Aminoglycosides; Anti-Bacterial Agents; Base Sequence; DNA Primers; DNA, Bacterial; Drug Resistance, Microbial; Escherichia coli; Genes, Bacterial; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Biosynthesis; Peptide Elongation Factor Tu; Peptides; Pyridones

Elongation factor Tu from Escherichia coli was mutated separately at positions Asp86 and Arg58, in order to shed light both on the GTPase mechanism of elongation factor Tu and on the binding of aminoacyl-tRNA. In addition, the binding of guanine nucleotides was investigated by determination of the dissociation and association rate constants. The results imply that Arg58 is unimportant for the intrinsic GTPase mechanism and the binding of guanine nucleotides, whereas it is strongly involved in the binding of aminoacyl-tRNA and of the ribosome. Asp86 appears to be essential for the regulation of guanine-nucleotide affinities, and it may also play a role in the intrinsic GTPase mechanism.

Topics: Anti-Bacterial Agents; Arginine; Aspartic Acid; Base Sequence; DNA Primers; Escherichia coli; GTP Phosphohydrolase-Linked Elongation Factors; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Models, Molecular; Molecular Sequence Data; Molecular Structure; Mutagenesis, Site-Directed; Peptide Elongation Factor Tu; Protein Binding; Pyridones; Recombinant Proteins; Ribosomes; RNA, Transfer; RNA, Transfer, Phe

A structural and functional understanding of resistance to the antibiotic kirromycin in Escherichia coli has been sought in order to shed new light on the functioning of the bacterial elongation factor Tu (EF-Tu), in particular its ability to act as a molecular switch. The mutant EF-Tu species G316D, A375T, A375V and Q124K, isolated by M13mp phage-mediated targeted mutagenesis, were studied. In this order the mutant EF-Tu species showed increasing resistance to the antibiotic as measured by poly(U)-directed poly(Phe) synthesis and intrinsic GTPase activities. The K'd values for kirromycin binding to mutant EF-Tu.GTP and EF-Tu.GDP increased in the same order. All mutation sites cluster in the interface of domains 1 and 3 of EF-Tu.GTP, not in that of EF-Tu.GDP. Evidence is presented that kirromycin binds to this interface of wild-type EF-Tu.GTP, thereby jamming the conformational switch of EF-Tu upon GTP hydrolysis. We conclude that the mutations result in two separate mechanisms of resistance to kirromycin. The first inhibits access of the antibiotic to its binding site on EF-Tu.GTP. A second mechanism exists on the ribosome, when mutant EF-Tu species release kirromycin and polypeptide chain elongation continues.

Topics: Anti-Bacterial Agents; Drug Resistance, Microbial; Escherichia coli; Guanosine Triphosphate; Models, Molecular; Mutation; Peptide Elongation Factor Tu; Protein Binding; Pyridones; Ribosomes; RNA, Bacterial; RNA, Transfer, Amino Acyl; Structure-Activity Relationship

Conformational transitions of Phe-tRNA(Phe) that take place during elongation factor Tu (EF-Tu)-dependent binding to the A site of Escherichia coli ribosomes were followed by transient fluorescence measurements. The fluorescence signal of proflavin replacing dihydrouracil at position 16 or 17 in yeast tRNA(Phe) was utilized to monitor changes of the conformation of the D loop. The ternary complex EF-Tu.GTP.Phe-TRNA(Phe)(Pf16/17) was purified by gel filtration. Upon binding of the complex to the A site of poly(U)-programmed, P-site-blocked ribosomes, the fluorescence changes in several steps. First, the rapid formation of an initial complex gives rise to a small fluorescence increase. Subsequent codon-anticodon recognition leads to a conformational rearrangement of the D loop of the tRNA that is reflected in a major fluorescence increase. Fluorescence-quenching data indicate an unfolding of the D loop in this state. The latter conformational state is short-lived, and the aminoacyl-tRNA refolds during the following rearrangement that occurs after GTP hydrolysis and accompanies the release of the aminoacyl-tRNA from EF-Tu.GDP and/or its accommodation in the A site. Further experiments show that the status of the P site influences the binding to the A site in that the two rearrangement steps are slowed down when the P site is unoccupied and even more so when it is occupied with the near-cognate tRNA(Leu2). In contrast, the occupancy of the E site has no influence on A-site binding, and vice versa, thus excluding any coupling between the two sites.

Topics: Anti-Bacterial Agents; Binding Sites; Buffers; Catalysis; Chromatography, Gel; Chromatography, High Pressure Liquid; Codon; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Guanosine Triphosphate; Hydrolysis; Nucleic Acid Conformation; Peptide Elongation Factor Tu; Poly U; Proflavine; Pyridones; Ribosomes; RNA, Transfer, Phe; Spectrometry, Fluorescence

The antibiotic kirromycin inhibits protein synthesis by binding to EF-Tu and preventing its release from the ribosome after GTP hydrolysis. We have isolated and sequenced a collection of kirromycin resistant tuf mutations and identified thirteen single amino acid substitutions at seven different sites in EF-Tu. These have been mapped onto the 3D structures of EF-Tu.GTP and EF-Tu.GDP. In the active GTP form of EF-Tu the mutations cluster on each side of the interface between domains I and III. We propose that this domain interface is the binding site for kirromycin.

Topics: Amino Acids; Anti-Bacterial Agents; Base Sequence; Binding Sites; DNA Mutational Analysis; Drug Resistance, Microbial; Guanosine Diphosphate; Guanosine Triphosphate; Models, Molecular; Molecular Sequence Data; Mutation; Peptide Elongation Factor Tu; Protein Conformation; Pyridones; Salmonella typhimurium

A remarkable positive cooperativity between the GTPase activities of EF-Tu and EF-G on empty ribosomes from Escherichia coli has been discovered. This cooperativity implies a decrease of the corresponding apparent KM values of the empty ribosome for either elongation factor: from more than 10 microM to 0.5 microM for EF-Tu.GTP by the addition of 0.25 microM EF-G and from 0.7 microM to 0.5 microM for EF-G.GTP by the addition of 3 microM EF-Tu. In a further analysis of this phenomenon, the effects of various specific antibiotics were studied: thiostrepton, fusidic acid, tetracycline, pulvomycin and kirromycin appeared to inhibit the synergistic effect, whereas streptomycin was found to stimulate it. Even in the present minimal system the ribosomes respond to the above-mentioned antibiotics in a way surprisingly similar to that in the coupled system with mRNA and tRNAs. The cooperativity seems not to be due to a simultaneous binding of the two elongation factors to the ribosome as revealed by studying the effects of fusidic acid and kirromycin, and by band-shift experiments by means of gel electrophoresis under non-denaturing conditions. Our experimental data and the kinetic analysis of alternative models provide evidence that EF-Tu.GTP and EF-G.GTP interact sequentially with empty ribosomes that oscillate between two different conformations, one for each elongation factor. Apparently, ribosomes have an intrinsic property for oscillation as normally observed during protein synthesis with a frequency paced by the events of tRNA binding and translocation.

Topics: Allosteric Regulation; Anti-Bacterial Agents; Electrophoresis; Escherichia coli; GTP Phosphohydrolase-Linked Elongation Factors; Guanosine Triphosphate; Kinetics; Models, Biological; Peptide Elongation Factor G; Peptide Elongation Factor Tu; Peptide Elongation Factors; Protein Binding; Pyridones; Ribosomes; Streptomycin

This paper reports the generation of Escherichia coli mutants resistant to pulvomycin. Together with targeted mutagenesis of the tufA gene, conditions were found to overcome membrane impermeability, thereby allowing the selection of three mutants harbouring elongation factor (EF)-Tu Arg230-->Cys, Arg333-->Cys or Thr334-->Ala which confer pulvomycin resistance. These mutations are clustered in the three-domain junction interface of the crystal structure of the GTP form of Thermus thermophilus EF-Tu. This result shares similarities with kirromycin resistance; kirromycin-resistant mutations cluster in the domain 1-3 interface. Since both interface regions are involved in the EF-Tu switch mechanism, we propose that pulvomycin and kirromycin both act by specifically disturbing the allosteric changes required for the switch from EF-Tu-GTP to EF-Tu-GDP. The three-domain junction changes dramatically in the switch to EF-Tu.GDP; in EF-Tu.GDP this region forms an open hole. Structural analysis of the mutation positions in EF-Tu.GTP indicated that the two most highly resistant mutants, R230C and R333C, are part of an electrostatic network involving numerous residues. All three mutations appear to destabilize the EF-Tu.GTP conformation. Genetic and protein characterizations show that sensitivity to pulvomycin is dominant over resistance. This appears to contradict the currently accepted model of protein synthesis inhibition by pulvomycin.

Topics: Aminoglycosides; Anti-Bacterial Agents; Binding Sites; Cell Membrane Permeability; Dose-Response Relationship, Drug; Drug Resistance, Microbial; Escherichia coli; Genes, Bacterial; Guanosine Triphosphate; Models, Biological; Models, Molecular; Molecular Conformation; Mutagenesis, Site-Directed; Peptide Biosynthesis; Peptide Elongation Factor Tu; Peptides; Protein Biosynthesis; Pyridones; RNA, Transfer, Amino Acyl; RNA, Transfer, Phe; Selection, Genetic; Structure-Activity Relationship

Elongation factor Tu (EF-Tu).GTP has the primary function of promoting the efficient and correct interaction of aminoacyl-tRNA with the ribosome. Very little is known about the elements in EF-Tu involved in this interaction. We describe a mutant form of EF-Tu, isolated in Salmonella typhimurium, that causes a severe defect in the interaction of the ternary complex with the ribosome. The mutation causes the substitution of Val for Gly-280 in domain II of EF-Tu. The in vivo growth and translation phenotypes of strains harboring this mutation are indistinguishable from those of strains in which the same tuf gene is insertionally inactivated. Viable cells are not obtained when the other tuf gene is inactivated, showing that the mutant EF-Tu alone cannot support cell growth. We have confirmed, by partial protein sequencing, that the mutant EF-Tu is present in the cells. In vitro analysis of the natural mixture of wild-type and mutant EF-Tu allows us to identify the major defect of this mutant. Our data shows that the EF-Tu is homogeneous and competent with respect to guanine nucleotide binding and exchange, stimulation of nucleotide exchange by EF-Ts, and ternary complex formation with aminoacyl-tRNA. However various measures of translational efficiency show a significant reduction, which is associated with a defective interaction between the ribosome and the mutant EF-Tu.GTP.aminoacyl-tRNA complex. In addition, the antibiotic kirromycin, which blocks translation by binding EF-Tu on the ribosome, fails to do so with this mutant EF-Tu, although it does form a complex with EF-Tu. Our results suggest that this region of domain II in EF-Tu has an important function and influences the binding of the ternary complex to the codon-programmed ribosome during protein synthesis. Models involving either a direct or an indirect effect of the mutation are discussed.

Topics: Anti-Bacterial Agents; Glycine; Guanosine Triphosphate; Hydrolysis; Kinetics; Macromolecular Substances; Models, Molecular; Peptide Elongation Factor Tu; Phenotype; Protein Biosynthesis; Protein Conformation; Pyridones; Ribosomes; RNA, Transfer, Amino Acyl; RNA, Transfer, Phe; Salmonella typhimurium; Valine

In a previous study (C. C. Hall, J. D. Watkins, and N. H. Georgopapadakou, Antimicrob. Agents Chemother. 33:322-325, 1989), the elongation factor Tu (EF-Tu) from Staphylococcus aureus was found to be insensitive to a series of kirromycin analogs which were inhibitory to the EF-Tu from Escherichia coli. In the present study, the EF-Tu from S. aureus was partially purified and characterized. Its apparent molecular mass was approximately 41,000 Da, and the enzyme copurified with EF-Ts (molecular mass, 34,000 Da). S. aureus EF-Tu differed from its E. coli counterpart in that it bound negligible amounts of [3H]GDP, in addition to being insensitive to pulvomycin and aurodox (50% inhibitory concentrations, approximately 100 and 1,000 microM, respectively, versus 2 and 0.2 microM, respectively, for E. coli). The results are consistent with the formation of a stable EF-Tu.EF-Ts complex that affects the interaction of EF-Tu with guanine nucleotides and inhibitors.

Topics: Adenosine Triphosphatases; Aminoglycosides; Anti-Bacterial Agents; Aurodox; Chromatography, DEAE-Cellulose; Drug Resistance, Microbial; Escherichia coli; Guanosine Diphosphate; Guanosine Triphosphate; Molecular Weight; Peptide Biosynthesis; Peptide Elongation Factor Tu; Peptides; Pyridones; Staphylococcus aureus

A simplified method for the separation of a kirromycin-sensitive tufB-encoded elongation factor Tu (EF-TuBs) from a kirromycin-resistant tufA product (EF-TuAr) was obtained by exploiting the specific increase of negative [corrected] charges induced by the antibiotic, resulting in a retarded elution of kirromycin-bound EF-TuBs on ionic chromatography. The kirromycin-free EF-TuBs is active in poly(Phe) synthesis and shows similar properties to EF-TuAsBs. As expected for these two distinct species, the dissociation of the EF-TuArBs.GTP complex in the presence of kirromycin shows a biphasic curve; in contrast, a monophasic GTP dissociation rate was found for a combination of two mutated EF-Tu species, EF-TuArBo, revealing the existence of intermolecular interactions. These observations prove for the first time the existence of cooperative phenomena between EF-Tu species in vitro, as suggested earlier by in vivo experiments.

Topics: Anti-Bacterial Agents; Chromatography, Liquid; Guanosine Triphosphate; Kinetics; Peptide Elongation Factor Tu; Pyridones

Intact, native EF-Tu, isolated using previously described methods and fully active in binding GTP, was never found to be fully active in binding aminoacyl-tRNA as judged by high performance liquid chromatography (HPLC) gel filtration and zone-interference gel-electrophoresis. In the presence of kirromycin, however, all these EF-Tu.GTP molecules bind aminoacyl-tRNA, although with a drastically reduced affinity. For the first time, the purification of milligram quantities of ternary complexes of EF-Tu.GTP and aminoacyl-tRNA, free of deacylated tRNA and inactive EF-Tu, has become possible using HPLC gel filtration. We also describe an alternative new method for the isolation of the ternary complexes by means of fractional extraction in the presence of polyethylene glycol. In the latter procedure, the solubility characteristics of the ternary complexes are highly reminiscent to those of free tRNA. Concentrated samples of EF-Tu.GMPPNP.aminoacyl-tRNA complexes show a high stability.

Topics: Electrophoresis, Polyacrylamide Gel; Escherichia coli; Guanosine Triphosphate; Guanylyl Imidodiphosphate; In Vitro Techniques; Macromolecular Substances; Peptide Elongation Factor Tu; Protein Binding; Pyridones; Ribonucleoproteins; RNA, Transfer, Amino Acyl; RNA, Transfer, Val

We have studied the interaction between EF-Tu-GDP or EF-Tu-GTP in complex with kirromycin or aurodox (N1-methylkirromycin) and aminoacyl-tRNA, N-acetylaminoacyl-tRNA, or deacylated tRNA. Three independent methods were used: zone-interference gel electrophoresis, GTPase stimulation, and fluorescence. All three methods revealed that kirromycin induces a severe drop in the stability of the complex of EF-Tu-GTP and aminoacyl-tRNA of about 3 orders of magnitude. The affinities of EF-Tu-kirromycin-GTP and EF-Tu-kirromycin-GDP for aa-tRNA were found to be of about the same order of magnitude. We conclude that kirromycin and related compounds do not induce a so-called GTP-like conformation of EF-Tu with respect to tRNA binding. The findings shed new light on the mechanism of action of the antibiotic during the elongation cycle. In contrast to indirect evidence previously obtained in our laboratory [Van Noort et al. (1982) EMBO J. 1, 1199-1205; Van Noort et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 71, 4910-4914], we were unable to demonstrate complexes of EF-Tu-aurodox-GTP/GDP with N-acetylaminoacyl-tRNA or deacylated tRNA by direct detection using zone-interference gel electrophoresis. Modification with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) decreases the affinity of EF-Tu-kirromycin-GTP for aminoacyl-tRNA, just like it does in the absence of the antibiotic.

Topics: Electrophoresis, Agar Gel; Escherichia coli; GTP Phosphohydrolase-Linked Elongation Factors; Guanosine Diphosphate; Guanosine Triphosphate; Peptide Elongation Factor Tu; Pyridones; RNA, Transfer, Amino Acyl; Spectrometry, Fluorescence; Substrate Specificity

The kirromycin-resistant EF-Tu mutant Aa, previously shown to be an antisuppressor for nonsense and missense suppressor tRNAs, has been characterised in a poly(U)-primed translation system in vitro. Two major defects were found in the function of the mutant. First, the dissociation constant for Aa binding to Phe-tRNA(Phe) was increased tenfold compared to wild-type EF-Tu. Second, kcat/Km for the interaction between the EF-Tu.GTP.aa-tRNA complex and the ribosome was decreased by the mutation to one third of its wild-type value. No differences were observed between mutant and wild-type factor in the regeneration of EF-Tu.GTP from EF-Tu.GDP via EF-Ts or in the mistranslation frequency by Leu-tRNA(4Leu). The relation between the in vitro results and the mutant phenotype in vivo is discussed.

Topics: Bacterial Proteins; Binding Sites; Drug Resistance; Escherichia coli; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Peptide Biosynthesis; Peptide Elongation Factor Tu; Peptide Elongation Factors; Phenotype; Protein Biosynthesis; Pyridones; Ribosomal Proteins; RNA, Transfer; RNA, Transfer, Amino Acid-Specific; Suppression, Genetic

The binding of Tyr-[AEDANS-s2C]tRNA(Tyr) (Tyr-tRNA(Tyr) modified at the penultimate cytidine residue with a thio group at position 2 of the pyrimidine ring, to which an N-(acetylaminoethyl)-5-naphthylamine-1-sulfonic acid fluorescence group is attached) to mutant elongation factor (EF)-Tu species from E. coli, EF-TuAR (Ala-375----Thr) and EF-TuBO (Gly-222----Asp), both complexed to GTP, was investigated in absence of kirromycin by measuring the change in fluorescence of the modified tRNA induced by complex formation. The calculated dissociation constant in the case of EF-TuAR is about 4 nM and in the case of EF-TuB0, about 1 nM. These values are higher than that of wild-type EF-Tu, which was 0.24 nM measured with the same system. The affinity between either EF-TuB0.kirromycin.GDP or EF-TuB0.kirromycin.GTP on the one hand, and a mixture of aminoacyl-tRNAs on the other, was measured with zone-interference gel electrophoresis. The dissociation constants are 20 microM and 7 microM, respectively, a factor of about two higher than in the case of wild-type EF-Tu.kirromycin. These findings provide a clue for the observed increase in translational errors in strains carrying the mutations. Furthermore, the experiments with EF-TuB0.kirromycin deepen our understanding of the effects of the B0 mutation on the kirromycin phenotype of the mutant cells concerned.

Topics: Anti-Bacterial Agents; Escherichia coli; Fluorescent Dyes; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Mutation; Naphthalenesulfonates; Peptide Elongation Factor Tu; Pyridones; RNA, Transfer, Tyr; Structure-Activity Relationship; Sulfhydryl Compounds

EF-Tu is often referred to as a model for guanine-nucleotide-binding regulatory proteins (G-proteins), since X-ray diffraction analysis of its GTP-binding domain shows a detailed location of the 'consensus' amino acid sequences involved in nucleotide binding. Fluoroaluminates are thought to mimick the gamma-phosphate in the GTPase centre on account of their activating effect on a variety of GTP binding proteins. In the case of EF-Tu, we could find no such effects on the basis of at least three independent functional assays. We did notice, however, complicating interactions between free nucleotides, fluoroaluminates and other ligands. By consequence, if indeed AlF4- behaves as a gamma-phosphate analogue in G-proteins, then EF-Tu must have a different GDP/GTP binding site, despite of the conserved consensus sequences.

Topics: Aluminum; Aluminum Chloride; Aluminum Compounds; Anti-Bacterial Agents; Binding Sites; Chlorides; Escherichia coli; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Peptide Elongation Factor Tu; Protein Conformation; Pyridones; Ribosomes; Sodium Fluoride; Vanadates; X-Ray Diffraction

The effect of guanine nucleotides and kirromycin on the conformation and stability of the chloroplast elongation factor Tu (EF-Tuchl) from Euglena gracilis has been investigated. Free EF-Tuchl is quite thermolabile but the protein is greatly stabilized by guanine nucleotides. The temperature dependence of the thermal inactivation of EF-Tuchl was used to calculate the amount of stabilization energy conferred by the guanine nucleotides. GDP increases the activation energy for the denaturation process by 77 kcal/mol while GTP increases the activation energy by 51 kcal/mol. The difference in heat stability of free EF-Tuchl and the EF-Tuchl.GDP complex was used to determine a dissociation constant of 1.3 x 10(-7) M at 37 degrees C. The temperature dependence of the dissociation constant allowed the calculation of a delta H degree obsd of -55 kcal/mol and a delta S degree obsd of -146 cal/(mol degree) for GDP binding to EF-Tuchl.EF-Tuchl was found to have a trypsin-sensitive region similar to that observed for Escherichia coli EF-Tu. This loop region was protected by GTP and kirromycin but not by GDP.

Topics: Animals; Anti-Bacterial Agents; Chloroplasts; Drug Stability; Euglena gracilis; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Peptide Elongation Factor Tu; Protein Conformation; Protein Denaturation; Pyridones; Thermodynamics

A new and general electrophoresis method is described for the determination of dissociation constants of weak macromolecular complexes in the range of 10(-6) to 10(-4) M. The method is based on the measurement of the migration distance of a macromolecular complex in rapid dynamic equilibrium as a function of the interacting ligand concentration in a surrounding zone. Special advantages of the method are: its high sensitivity (dependent on the autoradiography, immunoblotting or staining technique used), its speed (electrophoresis time 20 min), and the independence of the Kd determination on the sample concentration of macromolecules. The latter is of great value for labile macromolecules: unknown partial inactivation does not influence the measurement. Studying the interactions between elongation factor EF-Tu and tRNA from E. coli we found for EF-Tu.GTP.aurodox.aminocyl-tRNA a Kd of 3 microM and for EF-Tu.GDP.aurodox.aminoacyl-tRNA a Kd of 11 microM at 9 degrees C.

Topics: Anti-Bacterial Agents; Electrophoresis; Guanosine Triphosphate; Kinetics; Models, Theoretical; Nucleic Acids; Peptide Elongation Factor Tu; Protein Binding; Proteins; Pyridones; RNA, Transfer, Amino Acyl

The GTPase activity of purified EF-1 alpha from calf brain has been studied under various experimental conditions and compared with that of EF-Tu. EF-1 alpha displays a much higher GTPase turnover than EF-Tu in the absence of aminoacyl-tRNA (aa-tRNA) and ribosomes (intrinsic GTPase activity); this is due to the higher exchange rate between bound GDP and free GTP. Also the intrinsic GTPase of EF-1 alpha is enhanced by increasing the concentration of monovalent cations, K+ being more effective than NH+4. Differently from EF-Tu, aa-tRNA is much more active than ribosomes in stimulating the EF-1 alpha GTPase activity. However, ribosomes strongly reinforce the aa-tRNA effect. In the absence of aa-tRNA the rate-limiting step of the GTPase turnover appears to be the hydrolysis of GTP, whereas in its presence the GDP/GTP exchange reaction becomes rate-limiting, since addition of EF-1 beta enhances turnover GTPase activity. Kirromycin moderately inhibits the intrinsic GTPase of EF-1 alpha; this effect turns into stimulation when aa-tRNA is present. Addition of ribosomes abolishes any kirromycin effect. The inability of kirromycin to affect the EF-1 alpha/guanine-nucleotide interaction in the presence of ribosomes shows that, differently from EF-Tu, the EF-1 alpha X GDP/GTP exchange reaction takes place on the ribosome.

Topics: Animals; Brain Chemistry; Cattle; GTP Phosphohydrolase-Linked Elongation Factors; Guanosine Diphosphate; Guanosine Triphosphate; Ligands; Peptide Elongation Factor 1; Peptide Elongation Factors; Phosphoric Monoester Hydrolases; Poly U; Pyridones; Ribosomes; RNA, Messenger; RNA, Transfer, Amino Acyl

The properties of EF-1 alpha from calf brain have been investigated and compared with those of EF-Tu. EF-1 alpha binds GDP and GTP in a 1:1 stoichiometry, showing the same affinity for both nucleotides (K'd = 2-4 microM). EF-1 beta strongly enhances the dissociation rate of the EF-1 alpha X GDP complex and to a lesser extent of the EF-1 alpha X GTP complex. Aminoacyl-tRNA (aa-tRNA) stabilized EF-1 alpha X GTP much less efficiently than the EF-Tu X GTP complex. Unlike EF-Tu, EF-1 alpha sustains the binding of aa-tRNA to the ribosome also in the presence of GDP or in the absence of any nucleotide, though to a lesser degree than with GTP. Kirromycin enhances the dissociation rate of both EF-1 alpha X GTP and EF-1 alpha X GDP but especially that of the latter. This effect results in an increase of the exchange rate of the EF-1 alpha-bound nucleotide with free nucleotides. Although in this regard the effect of kirromycin mimics that of EF-1 beta, the antibiotic is incapable of increasing the EF-1 alpha X GDP/GTP exchange rate when aa-tRNA and ribosomes are present. Therefore, unlike EF-1 beta, kirromycin cannot enhance the rate of poly(Phe) synthesis. On the other hand, the failure of kirromycin to induce a GTP-like conformation of EF-1 alpha X GDP, as in the case of EF-Tu X GDP, explains its inability to inhibit peptide bond formation in the eukaryotic system.

Topics: Animals; Brain Chemistry; Cattle; Deoxycholic Acid; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Ligands; Peptide Elongation Factor 1; Peptide Elongation Factors; Protein Binding; Pyridones; RNA, Transfer, Amino Acyl

The interaction of the chloroplast elongation factor Tu (EF-Tuchl) from Euglena gracilis with guanine nucleotides and aminoacyl-tRNA has been investigated. The apparent dissociation constant at 37 degrees C for the EF-Tuchl X GDP complex is about 3 X 10(-7) M and for the EF-Tuchl X GTP complex, it is about 1 order of magnitude higher. The sulfhydryl modifying reagent N-ethylmaleimide severely inhibits the polymerization activity of Euglena EF-Tuchl. In the presence of N-ethylmaleimide, the dissociation constant for the modified EF-Tuchl X GDP complex is increased by an order of magnitude. Conversely, both GDP and GTP protect EF-Tuchl from the modification. The polymerization activity of EF-Tuchl is also sensitive to the antibiotic kirromycin. In the presence of kirromycin, the apparent dissociation constant for the EF-Tuchl X GTP complex is lowered 10-fold. The interaction of aminoacyl-tRNA with EF-Tuchl was investigated by examining the ability of EF-Tuchl to prevent the spontaneous hydrolysis of Phe-tRNA and by gel filtration chromatography. The binding of aminoacyl-tRNA to EF-Tuchl occurs only in the presence of GTP indicating the formation of the ternary complex EF-Tuchl X GTP X Phe-tRNA. The effect of kirromycin on the interaction was also investigated. In the presence of kirromycin, no interaction between EF-Tuchl and Phe-tRNA is observed, even in the presence of GTP.

Topics: Amino Acid Sequence; Chloroplasts; Ethylmaleimide; Euglena gracilis; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Magnesium; Peptide Elongation Factor Tu; Peptide Elongation Factors; Protein Conformation; Pyridones; RNA, Transfer, Amino Acyl

Pulvomycin and kirromycin, two antibiotics which inhibit protein biosynthesis in Escherichia coli by complex formation with the elongation factor Tu (EF-Tu), bind to different sites on the protein. While only one molecule of kirromycin can be bound to one molecule of EF-Tu, more than one molecule of pulvomycin interacts with a molecule of EF-Tu. This has been deduced from experiments in which the aminoacyl-tRNA binding and the GTPase activity of EF-Tu were measured in the presence of varying amounts of both antibiotics. These experiments are interpreted to mean that pulvomycin but not kirromycin can replace the other antibiotic in its respective site. Our conclusions are supported by circular dichroism spectroscopy.

Topics: Aminoglycosides; Anti-Bacterial Agents; Binding Sites; Circular Dichroism; Escherichia coli; Glycosides; GTP Phosphohydrolase-Linked Elongation Factors; Guanosine Triphosphate; Peptide Elongation Factor Tu; Peptide Elongation Factors; Pyridones; RNA, Transfer, Amino Acyl

The interaction of the polypeptide chain elongation factor Tu (EF-Tu) with the antibiotic kirromycin and tRNA has been studied by measuring the extent of protein modification with N-tosyl-L-phenylalanine chloromethylketone (TPCK) and N-ethylmaleimide (NEM). Kirromycin protects both EF-Tu.GDP and EF-Tu.GTP against modification with TPCK. Binding of aminoacyl-tRNA added at increasing concentrations to a solution of 40 microM EF-Tu.GDP.kirromycin complex re-exposes the TPCK target site on the protein. However, when the aminoacyl-tRNA concentration is raised beyond 20 microM, TPCK labeling drops again and is blocked completely at approximately 300 microM aminoacyl-tRNA. By contrast, addition of uncharged tRNA or N- acetylaminoacyl -tRNA enhances TPCK labeling of the protein over the entire tRNA concentration range studied. These data strongly suggest that kirromycin induces in EF-Tu.GDP an additional tRNA binding site that can bind uncharged tRNA, aminoacyl-tRNA, and N- acetylaminoacyl -tRNA. Support for this assumption is provided by measuring the modification of EF-Tu.GDP with the sulfhydryl reagent NEM. Moreover, NEM modification also indicates an additional tRNA binding site on EF-Tu.GTP.kirromycin, which could not be detected with TPCK. Mapping of the tryptic peptides of EF-Tu.GDP labeled with [14C]TPCK revealed only one target site for this agent, i.e., cysteine-81. Modification occurred at the same site in the presence and in the absence of kirromycin and uncharged tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Anti-Bacterial Agents; Binding Sites; Escherichia coli; Ethylmaleimide; Guanosine Diphosphate; Guanosine Triphosphate; Macromolecular Substances; Peptide Elongation Factor Tu; Peptide Elongation Factors; Protein Binding; Pyridones; RNA, Transfer; RNA, Transfer, Amino Acyl; Tosylphenylalanyl Chloromethyl Ketone

Properties of the elongation factor Tu from Lactobacillus brevis which is naturally insensitive to kirromycin are described. The protein is characterized by an unusual nucleotide-binding site with increased affinity for GTP and extreme heat stability. EF-Tu is sensitive to pulvomycin in the assay of polyphenylalanine synthesis. However, the failure of the protein to display pulvomycin-dependent GDP-binding and GTPase activity indicates that pulvomycin action in L. brevis differs from that in E. coli.

Topics: Aminoglycosides; Anti-Bacterial Agents; Drug Resistance, Microbial; Glycosides; Guanosine Triphosphate; Hot Temperature; Lactobacillus; Peptide Elongation Factor Tu; Peptide Elongation Factors; Pyridones

In the preceding article a mutant elongation factor Tu (EF-TuD2216) resistant to the action of kirromycin was found to display a spontaneous guanosine 5'-triphosphatase (GTPase) activity, i.e., in the absence of aminoacyl transfer ribonucleic acid (tRNA) and ribosome-messenger RNA. This is the first example of an Ef-Tu supporting GTPase activity in the absence of macromolecular effectors and/or kirromycin. In this study we show that this activity is elicited by increasing NH4+ concentrations. As additional effect, the mutation caused an increased affinity of EF-Tu for GTP. Ammonium dependence of the GTPase activity an increased affinity for GTP are two properties also found with wild-type EF-Tu in the presence of kirromycin [Fasano, O., Burns, W., Crechet, J.-B., Sander, G., & Parmeggiani, A. (1978) Eur. J. Biochem. 89, 557-565; Sander, G., Okonek, M., Crechet, J.-B., Ivell, R., Bocchini, V., & Parmeggiani, A. (1979) FEBS Lett. 98, 111-114]. Therefore, both binding of kirromycin to wild-type EF-Tu and acquisition of kirromycin resistance introduce functionally related modifications. Kirromycin at high concentrations (0.1 mM) does not interact with mutant EF-TuD2216.GDP but still does with EF-TuD2216.GTP in agreement with our previous finding that EF-Tu.GTP is the preferential target of the antibiotic in the wild type [Fasano, O., Bruns, W., Crechet, J.-B., Sander, G., & Parmeggiani, A. (1978) Eur. J. Biochem. 89, 557-565). The GTPase activity of mutant EF-Tu in the presence of aminoacyl-tRNA and ribosome.mRNA is much higher than with wild-type EF-Tu and also much less dependent on the presence of mRNA. Miscoding for leucine, measured as poly(U)-directed poly(phenyl-alanine/leucine) synthesis at increasing Mg2+ concentrations, is identical for both wild-type and mutant EF-Tu.

Topics: Ammonia; Anti-Bacterial Agents; Bacterial Proteins; Drug Resistance, Microbial; Escherichia coli; GTP Phosphohydrolases; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Mutation; Peptide Elongation Factor Tu; Phosphoric Monoester Hydrolases; Pyridones

Topics: Bacterial Proteins; Escherichia coli; GTP Phosphohydrolase-Linked Elongation Factors; Guanosine Triphosphate; Peptide Elongation Factor Tu; Peptide Elongation Factors; Phosphoric Monoester Hydrolases; Pyridones; Ribosomes; RNA, Transfer, Amino Acyl

Topics: Anti-Bacterial Agents; Bacterial Proteins; Escherichia coli; GTP Phosphohydrolases; Guanosine Triphosphate; Peptide Biosynthesis; Peptide Elongation Factor Tu; Peptides; Poly U; Pyridones; Ribosomes; RNA, Transfer, Amino Acyl; Structure-Activity Relationship

The activities of kirromycin oxime, aurodox 2,4-dinitrophenylhydrazone and four O-derivatives of aurodox have been compared to those of kirromycin (mocimycin) and its natural N-methyl analog aurodox in the in vitro system of E. coli. All synthetic derivatives were able to inhibit protein biosynthesis like the original antibiotics. Moreover, the analogs did promote all the effects of kirromycin on the reactions dependent on elongation factor Tu. From these results it can be concluded that the acidic hydroxyl and keto functions of kirromycin and aurodox are not directly involved in the action of the antibiotics on elongation factor Tu and can, thus, be chemically modified without loss of activity. In most cases, however, derivatization lowered the affinity of the antibiotic for elongation factor Tu. This suggests that the pyridone moiety of kirromycin and aurodox and the first part of its side chain should play a role in the association of these antibiotics with elongation factor Tu.

Topics: Anti-Bacterial Agents; Aurodox; Bacterial Proteins; Enzyme Activation; Escherichia coli; GTP Phosphohydrolase-Linked Elongation Factors; Guanosine Diphosphate; Guanosine Triphosphate; Peptide Elongation Factors; Pyridones; RNA, Transfer, Amino Acyl

Topics: Escherichia coli; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Magnetic Resonance Spectroscopy; Peptide Elongation Factors; Protein Binding; Pyridones; Water

In the accompanying paper we have shown that polypeptide synthesis sustained by the mutant elongation factor EF-TuBO is inhibited by kirromycin. Here we have searched for the primary site of inhibition in the elongation cycle. It is demonstrated that in the presence of the antibiotic EF-TuBO can form a complex with aminoacyl-tRNA and GTP and that the complex is able to bind to ribosomes programmed with poly(U). Like its wild-type counterpart, EF-TuBO . GDP can form a quaternary complex with aminoacyl-tRNA and kirromycin but, unlike the wild-type quaternary complex, the mutant complex fails to associate with the ribosome. This explains the recessive nature of the tuf B mutation in cells producing kirromycin-resistant EF-TuA and EF-TuBO. It also suggests a mechanism for the inhibition by kirromycin of EF-TuBO-dependent polypeptide synthesis.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Escherichia coli; Guanosine Triphosphate; Kinetics; Mutation; Peptide Chain Elongation, Translational; Peptide Elongation Factor Tu; Peptide Elongation Factors; Pyridones; Ribosomes; RNA, Transfer, Amino Acyl

Elongation factor Tu (EF-Tu) dependent GTP hydrolysis normally requires the presence of ribosomes and aminoacyl-tRNA (aa-tRNA). In the presence of the antibiotic kirromycin, the factor alone displays a GTPase activity that is enhanced by ribosomes and/or aa-tRNA [Wolf, H., Chinali, G., & Parmeggiani, A. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4910-4914]. Using this system, we have found the following: (1) the 50S ribosomal subunit can substitute the 70S ribosome; (2) the 50S CsCl core a, b, and c particles [Sander, G., Marsh, R. C., Voigt, J., & Parmeggiani, A. (1975) Biochemistry 14, 1805-1814], lacking an increasing number of proteins, can induce ca. 65, 45, and 25%, respectively, of the EF-Tu-kirromycin GTPase activity of control 50S subunits, in the presence of 30S subunits and aa-tRNA; (3) addition of proteins L7/L12 with L10, but not of proteins L7/L12 free from L10, restored the activity of all the 50S CsCl cores in the EF-Tu-kirromycin-dependent GTPase to 70-90% of the control; (4) proteins L7/L12, with or without contaminating L10, did not induce any EF-Tu-dependent GTPase activity, in contrast to a recent report [Donner, D., Villems, R., Liljas, A., & Kurland, C. G. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3192-3195], whether EF-Ts and/or kirromycin were present or not.

Topics: GTP Phosphohydrolase-Linked Elongation Factors; Guanosine Triphosphate; Kinetics; Peptide Elongation Factor Tu; Peptide Elongation Factors; Phosphoric Monoester Hydrolases; Pyridones; Ribosomal Proteins; Ribosomes

The protein synthesis elongation factors Tu and Ts are responsible for binding aminoacyl-transfer ribonucleic acid (RNA) to the ribosome. In addition, they perform an undefined function, as the EF-Tu.Ts complex, in the RNA phage RNA replicases. In an effort to obtain insight into these two apparently unrelated roles, we purified the elongation factors from Caulobacter crescentus and compared them to the analogous Escherichia coli polypeptides. Although most physical and functional characteristics were found to be similar, significant differences were found in the molecular weight of EF-Ts and relative affinities of guanine nucleotides, sensitivity to trypsin cleavage, and rate of heat denaturation of EF-Tu. The antibiotic kirromycin was active with EF-Tu from both bacterial species. When C. crescentus EF-Tu.Ts was substituted for the E. coli elongation factors in Q beta phage RNA replicase, an enzyme capable of apparently normal RNA synthetic activity was formed.

Topics: Anti-Bacterial Agents; Bacteria; Bacteriophages; GTP Phosphohydrolase-Linked Elongation Factors; Guanosine Diphosphate; Guanosine Triphosphate; Molecular Weight; Peptide Elongation Factors; Pyridones; RNA Nucleotidyltransferases; RNA-Dependent RNA Polymerase; Temperature; Trypsin

Topics: Ammonia; Anti-Bacterial Agents; Cations, Monovalent; Escherichia coli; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Peptide Elongation Factors; Pyridones; Ribosomes

Topics: Anti-Bacterial Agents; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Peptide Elongation Factors; Protein Binding; Pyridones; RNA, Transfer, Amino Acyl

The influence of kirromycin on the elongation factor Tu (EF-Tu) in its binary and ternary complexes was investigated. The equilibrium constant for the binding of the antibiotic to EF-Tu . GDP and EF-Tu . GTP was determined by circular dichroism titrations to be 4 x 10(6) M-1, and to EF-Tu . GTP . aa-tRNA by a combination of circular dichroism titrations and hydrolysis protection experiments to be 2 x 10(6) M-1. In the presence of kirromycin the binding of aminoacyl-tRNAs to EF-Tu . GTP is weakened by a factor of two. The antibiotic changes the conformation of the ternary complex in such a way that the aminoacyl moiety of the aminoacyl-tRNA is more accessible to the non-enzymatic hydrolysis. It is concluded that this structural alteration is responsible for the inhibitory action of the antibiotic.

Topics: Anti-Bacterial Agents; Circular Dichroism; Escherichia coli; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Peptide Elongation Factors; Pyridones

Topics: Anti-Bacterial Agents; Escherichia coli; GTP Phosphohydrolase-Linked Elongation Factors; Guanosine Triphosphate; Kinetics; Peptide Chain Elongation, Translational; Peptide Elongation Factors; Phenylalanine; Phosphoric Monoester Hydrolases; Protein Denaturation; Pyridones; Ribosomes; RNA, Transfer; Temperature

Kirromycin, a new inhibitor of protein synthesis, is shown to interfere with the peptide transfer reaction by acting on elongation factor Tu (EF-Tu). All the reactions associated with this elongation factor are affected. Formation of the EF-Tu.GTP complex is strongly stimulated. Peptide bond formation is prevented only when Phe-tRNA(Phe) is bound enzymatically to ribosomes, presumably because GTP hydrolysis associated with enzymatic binding of Phe-tRNA(Phe) is not followed by release of EF-Tu.GDP from the ribosome. This antibiotic also enables EF-Tu to catalyze the binding of Phe-tRNA(Phe) to the poly(U).ribosome complex even in the absence of GTP. EF-Tu activity in the GTPase reaction is dramatically affected by kirromycin: GTP hydrolysis, which normally requires ribosomes and aminoacyl-tRNA, takes place with the elongation factor alone. This GTPase shows the same K(m) for GTP as the one dependent on Phe-tRNA(Phe) and ribosomes in the absence of the antibiotic. Ribosomes and Phe-tRNA(Phe), but not tRNA(Phe) or Ac-Phe-tRNA(Phe), stimulate the kirromycin-induced EF-Tu GTPase. These results indicate that the catalytic center of EF-Tu GTPase that is dependent upon aminoacyl-tRNA and ribosomes is primarily located on the elongation factor. In conclusion, kirromycin can substitute for GTP, aminoacyl-tRNA, or ribosomes in various reactions involving EF-Tu, apparently by affecting the allosteric controls between the sites on the EF-Tu molecule interacting with these components.

Topics: Anti-Bacterial Agents; Carbon Radioisotopes; Depression, Chemical; Escherichia coli; Furans; Guanosine; Guanosine Triphosphate; Peptide Chain Elongation, Translational; Peptide Elongation Factors; Phenylalanine; Phosphoric Monoester Hydrolases; Phosphorus Radioisotopes; Pyridones; Ribosomes; RNA, Transfer; Tritium