GE2270A-resistant mutations in elongation factor Tu allow productive aminoacyl-tRNA binding to EF-Tu.GTP.GE2270A complexes

The antibiotic GE2270A prevents stable complex formation between elongation factor Tu (EF-Tu) and aminoacyl-tRNA (aatRNA). In Escherichia coli we characterized two mutant EF-Tu species with either G257S or G275A that lead to high GE2270A resistance in poly(Phe) synthesis, which at least partially explains the high resistance of EF-Tu1 from GE2270A producer Planobispora rosea to its own antibiotic. Both E. coli mutants were unexpectedly found to bind GE2270A nearly as well as wild-type (wt) EF-Tu in their GTP-bound conformations. Both G257S and G275A are in or near the binding site for the 3' end of aatRNA. The G257S mutation causes a 2.5-fold increase in affinity for aatRNA, whereas G275A causes a 40-fold decrease. In the presence of GE2270A, wt EF-Tu shows a drop in aatRNA affinity of at least four orders of magnitude. EF-Tu[G275S] and EF-Tu[G275A] curtail this drop to about two or one order, respectively. It thus appears that the resistance mutations do not prevent GE2270A from binding to EF-Tu.GTP and that the mutant EF-Tus may accommodate GE2270A and aatRNA simultaneously. Interestingly, in their GDP-bound conformations the mutant EF-Tus have much less affinity for GE2270A than wt EF-Tu. The latter is explained by a recent crystal structure of the EF-Tu.GDP.GE2270A complex, which predicts direct steric problems between GE2270A and the mutated G257S or G275A. These mutations may cause a dislocation of GE2270A in complex with GTP-bound EF-Tu, which then no longer prevents aatRNA binding as in the wt situation. Altogether, the data lead to the following novel resistance scenario. Upon arrival of the mutant EF-Tu.GTP.GE2270.aatRNA complex at the ribosomal A-site, the GTPase centre is triggered. The affinities of aatRNA and GE2270A for the GDP-bound EF-Tu are negligible; the former stays at the A-site for subsequent interaction with the peptidyltransferase centre and the latter two dissociate from the ribosome.     

Zone-interference gel electrophoresis: a new method for studying weak protein-nucleic acid complexes under native equilibrium conditions.

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.     

Mutant EF-Tu species reveal novel features of the enacyloxin IIa inhibition mechanism on the ribosome

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.     

Interaction of mitochondrial elongation factor Tu with aminoacyl-tRNA and elongation factor Ts

Elongation factor (EF) Tu promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. This process requires the formation of a ternary complex (EF-Tu.GTP.aa-tRNA). EF-Tu is released from the ribosome as an EF-Tu.GDP complex. Exchange of GDP for GTP is carried out through the formation of a complex with EF-Ts (EF-Tu.Ts). Mammalian mitochondrial EF-Tu (EF-Tu(mt)) differs from the corresponding prokaryotic factors in having a much lower affinity for guanine nucleotides. To further understand the EF-Tu(mt) subcycle, the dissociation constants for the release of aa-tRNA from the ternary complex (K(tRNA)) and for the dissociation of the EF-Tu.Ts(mt) complex (K(Ts)) were investigated. The equilibrium dissociation constant for the ternary complex was 18 +/- 4 nm, which is close to that observed in the prokaryotic system. The kinetic dissociation rate constant for the ternary complex was 7.3 x 10(-)(4) s(-)(1), which is essentially equivalent to that observed for the ternary complex in Escherichia coli. The binding of EF-Tu(mt) to EF-Ts(mt) is mutually exclusive with the formation of the ternary complex. K(Ts) was determined by quantifying the effects of increasing concentrations of EF-Ts(mt) on the amount of ternary complex formed with EF-Tu(mt). The value obtained for K(Ts) (5.5 +/- 1.3 nm) is comparable to the value of K(tRNA).     

The G222D mutation in elongation factor Tu inhibits the codon-induced conformational changes leading to GTPase activation on the ribosome.

Elongation factor Tu (EF-Tu) from Escherichia coli carrying the mutation G222D is unable to hydrolyze GTP on the ribosome and to sustain polypeptide synthesis at near physiological Mg2+ concentration, although the interactions with guanine nucleotides and aminoacyl-tRNA are not changed significantly. GTPase and polypeptide synthesis activities are restored by increasing the Mg2+ concentration. Here we report a pre-steady-state kinetic study of the binding of the ternary complexes of wild-type and mutant EF-Tu with Phe-tRNA(Phe) and GTP to the A site of poly(U)-programed ribosomes. The kinetic parameters of initial binding to the ribosome and subsequent codon-anticodon interaction are similar for mutant and wild-type EF-Tu, independent of the Mg2+ concentration, suggesting that the initial interaction with the ribosome is not affected by the mutation. Codon recognition following initial binding is also not affected by the mutation. The main effect of the G222D mutation is the inhibition, at low Mg2+ concentration, of codon-induced structural transitions of the tRNA and, in particular, their transmission to EF-Tu that precedes GTP hydrolysis and the subsequent steps of A-site binding. Increasing the Mg2+ concentration to 10 mM restores the complete reaction sequence of A-site binding at close to wild-type rates. The inhibition of the structural transitions is probably due to the interference of the negative charge introduced by the mutation with negative charges either of the 3' terminus of the tRNA, bound in the vicinity of the mutated amino acid in domain 2 of EF-Tu, or of the ribosome. Increasing the Mg2+ concentration appears to overcome the inhibition by screening the negative charges.     

The elongation factor Tu.kirromycin complex has two binding sites for tRNA molecules

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)     

The complex formation between Escherichia coli aminoacyl-tRNA, elongation factor Tu and GTP. The effect of the side-chain of the amino acid linked to tRNA

The interaction between Escherichia coli aminoacyl-tRNAs and elongation factor Tu (EF-Tu) x GTP was examined. Ternary complex formation with Phe-tRNAPhe and Lys-tRNALys was compared to that with the respective misaminoacylated Tyr-tRNAPhe and Phe-tRNALys. There was no pronounced difference in the efficiency of aminoacyl-tRNA x EF-Tu x GTP complex formation between Phe-tRNAPhe and Tyr-tRNAPhe. However, Phe-tRNALys was bound preferentially to EF-Tu x GTP as compared to Lys-tRNALys. This was shown by the ability of EF-Tu x GTP to prevent the hydrolysis of the aminoacyl ester linkage of the aminoacyl-tRNA species. Furthermore, gel filtration of ternary complexes revealed that the complex formed with the misaminoacylated tRNALys was also more stable than the one formed with the correctly aminoacylated tRNALys. Both misaminoacylated aminoacyl-tRNA species could participate in the ribosomal peptide elongation reaction. Poly(U)-directed synthesis of poly(Tyr) using Tyr-tRNAPhe occurred to a comparable extent as the synthesis of poly(Phe) with Phe-tRNAPhe. In the translation of poly(A) using native Lys-tRNALys, poly(Lys) reached a lower level than poly(Phe) when Phe-tRNALys was used. It was concluded that the side-chain of the amino acid linked to a tRNA affects the efficiency of the aminoacyl-tRNA x EF-Tu x GTP ternary complex formation.     

Efficient separation of Thermus aquaticus EF-Tu functional complexes

A new method for fast separation of the main functional complexes of the elongation factor Tu from Thermus aquaticus has been developed. Binary complexes EF-Tu * GDP and EF-Tu * GDPNP as well as the ternary complex EF-Tu * GDPNP * Leu approximately tRNA were separated from each other by means of HPLC on a hydrophobic sorbent TSK-Gel Phenyl 5PW in a reverse gradient of ammonium sulfate. This technique is suitable for monitoring EF-Tu activity, characterisation of the ratio between different EF-Tu forms in cell extracts, and isolation of individual EF-Tu complexes for structural and functional investigations. In order to illustrate the potentials of the method, we used HPLC on a TSK-Gel Phenyl 5PW matrix to determine the ratio between affinities of GDP and GDPNP for EF-Tu. We found that K(a)(GDP) is about 27 times higher than K(a)(GDPNP) at 37 degrees C, the value being close to the one reported for Thermus thermophilus EF-Tu.     

A single amino acid substitution in elongation factor Tu disrupts interaction between the ternary complex and the ribosome.

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.     

Isolation and stability of ternary complexes of elongation factor Tu, GTP and aminoacyl-tRNA.

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.     

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.     

Common location of determinants in initiator transfer RNAs for initiator-elongator discrimination in bacteria and in eukaryotes

Initiator tRNAs are used exclusively for initiation of protein synthesis and not for elongation. We show that both Escherichia coli and eukaryotic initiator tRNAs have negative determinants, at the same positions, that block their activity in elongation. The primary negative determinant in E. coli initiator tRNA is the C1xA72 mismatch at the end of the acceptor stem. The primary negative determinant in eukaryotic initiator tRNAs is located in the TPsiC stem, whereas a secondary negative determinant is the A1:U72 base pair at the end of the acceptor stem. Here we show that E. coli initiator tRNA also has a secondary negative determinant for elongation and that it is the U50.G64 wobble base pair, located at the same position in the TPsiC stem as the primary negative determinant in eukaryotic initiator tRNAs. Mutation of the U50.G64 wobble base pair to C50:G64 or U50:A64 base pairs increases the in vivo amber suppressor activity of initiator tRNA mutants that have changes in the acceptor stem and in the anticodon sequence necessary for amber suppressor activity. Binding assays of the mutant aminoacyl-tRNAs carrying the C50 and A64 changes to the elongation factor EF-Tu.GTP show marginally higher affinity of the C50 and A64 mutant tRNAs and increased stability of the EF-Tu.GTP. aminoacyl-tRNA ternary complexes. Other results show a large effect of the amino acid attached to a tRNA, glutamine versus methionine, on the binding affinity toward EF-Tu.GTP and on the stability of the EF-Tu.GTP.aminoacyl-tRNA ternary complex.     

Base 2661 in Escherichia coli 23S rRNA influences the binding of elongation factor Tu during protein synthesis in vivo.

The binding of the EF-Tu.GTP.aminoacyl-tRNA ternary complex (EF, elongation factor) to the ribosome is known to be strengthened by a 2661G-to-C mutation in 23S ribosomal RNA, whereas the binding to normal ribosomes is weakened if the factor is in an appropriate mutant form (Aa). In this report we describe the mutual effects by the 2661C alteration in 23S rRNA and EF-Tu(Aa) on bacterial viability and translation efficiency in strains with normal or mutationally altered ribosomes. The rrnB(2661C) allele on a multicopy plasmid was introduced by transformation into Escherichia coli K-12 strains, harbouring either the wild-type or the mutant gene (tufA) for EF-Tu as well as normal or mutant ribosomal protein S12 (rpsL). Together with wild-type EF-Tu, the 2661C mutant ribosomes decreased the translation elongation rate in a rpsL+ strain or a non-restrictive rpsL224 strain. This reduction was not seen in strains which harbored EF-Tu(Aa) instead of EF-Tu(As) (As, wild-type form). Nonsense codon suppression by tyrT(Su3) suppressor tRNA was reduced by 2661C in a rpsL224 strain in the presence of EF-Tu(As) but not in the presence of EF-Tu(Aa). The lethal effect obtained by the combination of 2661C and a restrictive ribosomal protein S12 mutation (rpsL282) disappeared if EF-Tu(As) was replaced by EF-Tu(Aa) in the strain. In such a viable strain, 2661C had no effect on either the translation elongation rate or nonsense codon suppression. Our data suggest that the G base at position 2661 in 23S rRNA is important for binding of EF-Tu during protein synthesis in vivo. The interaction between this base and EF-Tu is strongly influenced by the structure of ribosomal protein S12.     

Altered regulation of the guanosine 5'-triphosphate activity in a kirromycin-resistant elongation factor Tu

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.     

A study of a mutant elongation factor properties of E. coli HAK88 and its mutant elongation factor Tu.

Summary The E. coli chromosome contains two genes for elongation factor Tu, tufA (near the fusidic acid resistance marker) and tufB (near the rifampicin resistance marker). It has been discovered that the mutant E. coli K12 strain HAK88 bears a mutation in the tufB gene, which leads to the synthesis of a protein of increased acidity. To determine whether the mutation has altered the protein's function in peptide chain elongation, we have compared the reactivities of normal tufA EF-Tu and mutant tufB EF-Tu (purified together from HAK88) with the components of the AA-tRNA binding cycle. Normal tufA EF-Tu and mutant tufB EF-Tu are indistinguishable in their affinities for GDP, EF-Ts, and phe-tRNA, and differ only slightly in their affinities for ribosomes. Coupled with the results of a separate study showing the similarity of the normal tufA and tufB gene products, these experiments demonstrate that the mutation has not altered the function of tufB EF-Tu in peptide chain elongation. Contrary to the original report (Kuwano et al., 1974; J. Mol. Biol. 86, 689–698) the HAK88 strains we have examined no longer possess a temperature-sensitive EF-Ts. The growth rates of HAK88 strains resemble the parent HAK8 strain in their lack of tRNA dependence but unlike HAK8 show varying degrees of temperature sensitivity. We conclude that HAK88 contains a physically altered but functionally intact tufB EF-Tu. The mutation in tufB should be valuable for studying in vivo the control of expression of the genes for EF-Tu.     

The affinity of elongation factor Tu for an aminoacyl-tRNA is modulated by the esterified amino acid

When different mutations were introduced into the anticodon loop and at position 73 of YFA2, a derivative of yeast tRNA(Phe), a single tRNA body was misacylated with 13 different amino acids. The affinities of these misacylated tRNAs for Thermus thermophilus elongation factor Tu (EF-Tu).GTP were determined using a ribonuclease protection assay. A range of 2.5 kcal/mol in the binding energies was observed, clearly demonstrating that EF-Tu specifically recognizes the side chain of the esterified amino acid. Furthermore, this specificity can be altered by introducing a mutation in the amino acid binding pocket on the surface of EF-Tu. Also, when discussed in conjunction with the previously determined specificity of EF-Tu for the tRNA body, these experiments further demonstrate that EF-Tu uses thermodynamic compensation to bind cognate aminoacyl-tRNAs similarly.     

Translational frameshifts induced by mutant species of the polypeptide chain elongation factor Tu of Escherichia coli

Translational frameshifts, both +1 and -1, are promoted by mutations in tufA and tufB, the two genes encoding the polypeptide chain elongation factor (EF) Tu of Escherichia coli. Strains harboring the mutant EF-Tu(Ala375----Thr) encoded by either tufA or tufB or by both, display a linear relationship between the frequency of frameshifting and the concentration of mutant EF-Tu, relative to the total amount of EF-Tu. A second mutant species, EF-TuB(Gly222----Asp), also promotes frameshifting. The frequency is strikingly enhanced by the combined action of EF-TuA(Ala375----Thr) and EF-TuB(Gly222----Asp) and exceeds by far the total contribution of the two mutant EF-Tus studied separately. These observations raise the question whether the formation of each peptide bond under conditions that no frameshifting occurs also requires the combined action of two EF-Tu molecules, in this case not differing functionally.     

The 51-63 base pair of tRNA confers specificity for binding by EF-Tu

Elongation factor Tu (EF-Tu) exhibits significant specificity for the different elongator tRNA bodies in order to offset its variable affinity to the esterified amino acid. Three X-ray cocrystal structures reveal that while most of the contacts with the protein involve the phosphodiester backbone of tRNA, a single hydrogen bond is observed between the Glu390 and the amino group of a guanine in the 51-63 base pair in the T-stem of tRNA. Here we show that the Glu390Ala mutation of Thermus thermophilus EF-Tu selectively destabilizes binding of those tRNAs containing a guanine at either position 51 or 63 and that mutagenesis of the 51-63 base pair in several tRNAs modulates their binding affinities to EF-Tu. A comparison of Escherichia coli tRNA sequences suggests that this specificity mechanism is conserved across the bacterial domain. While this contact is an important specificity determinant, it is clear that others remain to be identified.     

Mechanism of dihydroneopterin aldolase: functional roles of the conserved active site glutamate and lysine residues

Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) in the folate biosynthetic pathway. There are four conserved active site residues at the active site, E22, Y54, E74, and K100 in Staphylococcus aureus DHNA (SaDHNA), corresponding to E21, Y53, E73, and K98, respectively, in Escherichia coli DHNA (EcDHNA). The functional roles of the conserved glutamate and lysine residues have been investigated by site-directed mutagenesis in this work. E22 and E74 of SaDHNA and E21, E73, and K98 of EcDHNA were replaced with alanine. K100 of SaDHNA was replaced with alanine and glutamine. The mutant proteins were characterized by equilibrium binding, stopped-flow binding, and steady-state kinetic analyses. For SaDHNA, none of the mutations except E74A caused dramatic changes in the affinities of the enzyme for the substrate or product analogues or the rate constants. The Kd values for SaE74A were estimated to be >3000 microM, suggesting that the Kd values of the mutant are at least 100 times those of the wild-type enzyme. For EcDHNA, the E73A mutation increased the Kd values for the substrate or product analogues neopterin (MP), monapterin (NP), and 6-hydroxypterin (HPO) by factors of 340, 160, and 5600, respectively, relative to those of the wild-type enzyme. The K98A mutation increased the Kd values for NP, MP, and HPO by factors of 14, 3.6, and 230, respectively. The E21A mutation increased the Kd values for NP and HPO by factors of 2.2 and 42, respectively, but decreased the Kd value for MP by a factor of 3.3. The E22 (E21) and K100 (K98) mutations decreased the kcat values by factors of 1.3-2 x 10(4). The E74 (E73) mutation decreased in the kcat values by factors of approximately 10. The results suggested that E74 of SaDHNA and E73 of EcDHNA are important for substrate binding, but their roles in catalysis are minor. In contrast, E22 and K100 of SaDHNA are important for catalysis, but their roles in substrate binding are minor. On the other hand, E21 and K98 of EcDHNA are important for both substrate binding and catalysis.