Identification of Novel RNA-Protein Contact in Complex of Ribosomal Protein S7 and 3'-Terminal Fragment of 16S rRNA in E. coli.

For prokaryotes in vitro, 16S rRNA and 20 ribosomal proteins are capable of hierarchical self- assembly yielding a 30S ribosomal subunit. The self-assembly is initiated by interactions between 16S rRNA and three key ribosomal proteins: S4, S8, and S7. These proteins also have a regulatory function in the translation of their polycistronic operons recognizing a specific region of mRNA. Therefore, studying the RNA-protein interactions within binary complexes is obligatory for understanding ribosome biogenesis. The non-conventional RNA-protein contact within the binary complex of recombinant ribosomal protein S7 and its 16S rRNA binding site (236 nucleotides) was identified. UV-induced RNA-protein cross-links revealed that S7 cross-links to nucleotide U1321 of 16S rRNA. The careful consideration of the published RNA- protein cross-links for protein S7 within the 30S subunit and their correlation with the X-ray data for the 30S subunit have been performed. The RNA - protein cross-link within the binary complex identified in this study is not the same as the previously found cross-links for a subunit both in a solution, and in acrystal. The structure of the binary RNA-protein complex formed at the initial steps of self-assembly of the small subunit appears to be rearranged during the formation of the final structure of the subunit.

Selection of random RNA fragments as method for searching for a site of regulation of translation of E. coli streptomycin mRNA by ribosomal protein S7.

In E. coli cells ribosomal small subunit biogenesis is regulated by RNA-protein interactions involving protein S7. S7 initiates the subunit assembly interacting with 16S rRNA. During shift-down of rRNA synthesis level, free S7 inhibits self-translation by interacting with 96 nucleotides long specific region of streptomycin (str) mRNA between cistrons S12 and S7 (intercistron). Many bacteria do not have the extended intercistron challenging development of specific approaches for searching putative mRNA regulatory regions, which are able to interact with proteins. The paper describes application of SERF approach (Selection of Random RNA Fragments) to reveal regulatory regions of str mRNA. Set of random DNA fragments has been generated from str operon by random hydrolysis and then transcribed into RNA; the fragments being able to bind protein S7 (serfamers) have been selected by iterative rounds. S7 binds to single serfamer, 109 nucleotide long (RNA109), derived from the intercistron. After multiple copying and selection, the intercistronic mutant (RNA109) has been isolated; it has enhanced affinity to S7. RNA109 binds to the protein better than authentic intercistronic str mRNA; apparent dissociation constants are 26 +/- 5 and 60 +/- 8 nM, respectively. Location of S7 binding site on the mRNA, as well as putative mode of regulation of coupled translation of S12 and S7 cistrons have been hypothesized.

Quality control of the elongation step of protein synthesis by tmRNP.

Trans-translation is a quality-control process, activated upon premature termination of protein elongation, which recycles stalled ribosomes and degrades incomplete polypeptides. These functions are facilitated by transfer-messenger RNA (tmRNA, also called 10Sa RNA or SsrA RNA), a small stable RNA molecule encoded by the SsrA gene found in bacteria, chloroplasts and mitochondria. Most tmRNAs consist of a tRNA- and an mRNA-like domain connected by up to four pseudoknots. Comparative sequence analysis provided the first insight into tmRNA secondary and three-dimensional structure. Studies of the E. coli tmRNA in vitro and in vivo demonstrated that tmRNA functions as a ribonucleoprotein (RNP) complex with elongation factor Tu (EF-Tu), protein SmpB and ribosomal protein S1. The tRNA-like and mRNA-like activities of tmRNA mark prematurely terminated proteins for degradation by attaching to their C-termini peptide tags, which are recognized by numerous proteases. Studies aimed at understanding the details of the molecular mechanisms of trans-translation are ongoing.

Simultaneous and functional binding of SmpB and EF-Tu-TP to the alanyl acceptor arm of tmRNA.

Transfer-messenger RNA (tmRNA) mimics functions of aminoacyl-tRNA and mRNA, subsequently, when rescuing stalled ribosomes on a 3' truncated mRNA without stop codon in bacteria. In addition, this mechanism marks prematurely terminated proteins by a C-terminal peptide tag as a signal for degradation by specific cellular proteases. For Escherichia coli, previous studies on initial steps of this "trans-translation" mechanism revealed that tmRNA alanylation by Ala-tRNA synthetase and binding of Ala-tmRNA by EF-Tu-GTP for subsequent delivery to stalled ribosomes are inefficient when compared to analogous reactions with canonical tRNA(Ala). In other studies, protein SmpB and ribosomal protein S1 appeared to bind directly to tmRNA and to be indispensable for trans-translation. Here, we have searched for additional and synergistic effects of the latter two on tmRNA alanylation and its subsequent binding to EF-Tu-GTP. Kinetic analysis of functioning combined with band-shift experiments and structural probing demonstrate, that tmRNA may indeed form a multimeric complex with SmpB, S1 and EF-Tu-GTP, which leads to a considerably enhanced efficiency of the initial steps of trans-translation. Whereas S1 binds to the mRNA region of tmRNA, we have found that SmpB and EF-Tu both interact with its acceptor arm region. Interaction with SmpB and EF-Tu was also observed at the acceptor arm of Ala-tRNA(Ala), but there the alanylation efficiency was inhibited rather than stimulated by SmpB. Therefore, SmpB may function as an essential modulator of the tRNA-like acceptor arm of tmRNA during its successive steps in trans-translation.

The variant tuf3 gene of Streptomyces coelicolor A3(2) encodes a real elongation factor Tu, as shown in a novel Streptomyces in vitro translation system.

In Streptomyces coelicolor, the regular and abundant elongation factor (EF)-Tu1 is encoded by tuf1, while the actual function of the highly divergent tuf3 gene product is not yet known. Expression of the latter could so far only be detected on the transcriptional level under stress conditions. In this paper we demonstrate the presence of low levels of EF-Tu3 in strains of the J1501 lineage. Enhanced expression was observed for J1501 glkA and relA deletion mutants, which lack glucose kinase and ribosome-bound ppGpp synthetase, respectively. To assess the putative translational capacities of EF-Tu3, a novel Streptomyces in vitro translation assay was designed, based on the full elimination by Ni2+ affinity adsorption of chromosomally encoded (His)6-tagged EF-Tu1 from a S. coelicolor cell-free extract. Translational activity of this system is totally dependent on the addition of purified EF-Tu species or on the presence of an additional elongation factor Tu in the extract, e.g. encoded by a plasmid-borne tuf gene. Using this EF-Tu-dependent translation system, we have established that S. coelicolor EF-Tu3 has translational capacities despite its striking deviation from the common prokaryotic EF-Tu sequence at positions involved in either aminoacyl-tRNA binding or interaction with the guanine-nucleotide exchange factor EF-Ts.

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.

ssgA is essential for sporulation of Streptomyces coelicolor A3(2) and affects hyphal development by stimulating septum formation.

The role of ssgA in cell division and development of streptomycetes was analyzed. An ssgA null mutant of Streptomyces coelicolor produced aerial hyphae but failed to sporulate, and ssgA can therefore be regarded as a novel whi gene. In addition to the morphological changes, antibiotic production was also disturbed, with strongly reduced actinorhodin production. These defects could be complemented by plasmid-borne ssgA. In the wild-type strain, transcription of ssgA was induced by nutritional shift-down and was shown to be linked to that of the upstream-located gene ssgR, which belongs to the family of iclR-type transcriptional regulator genes. Analysis of mycelium harvested from liquid-grown cultures by transmission electron microscopy showed that septum formation had strongly increased in ssgA-overexpressing strains in comparison to wild-type S. coelicolor and that spore-like compartments were produced at high frequency. Furthermore, the hyphae were significantly wider and contained irregular and often extremely thick septa. These data underline the important role for ssgA in Streptomyces cell division.

Kinetic parameters for tmRNA binding to alanyl-tRNA synthetase and elongation factor Tu from Escherichia coli.

Aminoacylation and transportation of tmRNA to stalled ribosomes constitute prerequisite steps for trans-translation, a process facilitating the release of stalled ribosomes from 3' ends of truncated mRNAs and the degradation of incompletely synthesized proteins. Kinetic analysis of the aminoacylation of tmRNA indicates that tmRNA has both a lower affinity and a lower turnover number than cognate tRNA(Ala) for alanyl-tRNA synthetase, resulting in a 75-fold lower k(cat)/K(M) value. The association rate constant of Ala-tmRNA for elongation factor Tu in complex with GTP is about 150-fold lower than that of Ala-tRNA(Ala), whereas its dissocation rate constant is about 5-fold lower. These observations can be interpreted to suggest that additional factors facilitate tmRNA binding to ribosomes.

The effect of mutations in EF-Tu on its affinity for tRNA as measured by two novel and independent methods of general applicability.

Elongation factor Tu is essential for binding and a correct delivery of aminoacyl-tRNA during protein biosynthesis. For a good characterization of its interaction with tRNA in terms of structure-function relationship, determinations of kinetic equilibrium parameters are of great value. We describe two novel methods for that purpose. One method is based on EF-Tu protection of the tRNA 3' acceptor end against RNase A cleavage and yields the Kd value together with the corresponding dissociation and association rate constants from one single set of experiments. The other is a rapid method for screening relative affinities of mutant EF-Tus for tRNA. It is based on competition between EF-Tu species with and without a (His)6 extension for the same aminoacyl-tRNA and yields a relative Kd value. The method can be of general importance for the measuring of ligand affinities of all sorts of His-tagged proteins. Both methods are illustrated by their application in the analysis of mutant EF-Tus with changed interactions with tRNA and antibiotics. Raising the assay temperature from 4 to 37 degrees C causes a 30-fold increase of Kd for EF-Tu x GTP x Phe-tRNA complexes. The mutation K237E leads to rapid inactivation at the latter temperature. A parallel is found between the order of increasing Kd values for EF-Tus with mutation G316D, A375T and Q124K, respectively, and their order of increasing resistance to kirromycin.

Effects of increased and deregulated expression of cell division genes on the morphology and on antibiotic production of streptomycetes.

This paper describes the effects of increased expression of the cell division genes ftsZ, ftsQ, and ssgA on the development of both solid- and liquid-grown mycelium of Streptomyces coelicolor and Streptomyces lividans. Over-expression of ftsZ in S. coelicolor M145 inhibited aerial mycelium formation and blocked sporulation. Such deficient sporulation was also observed for the ftsZ mutant. Over-expression of ftsZ also inhibited morphological differentiation in S. lividans 1326, although aerial mycelium formation was less reduced. Furthermore, antibiotic production was increased in both strains, and in particular the otherwise dormant actinorhodin biosynthesis cluster of S. lividans was activated in liquid- and solid-grown cultures. No significant alterations were observed when the gene dosage of ftsQ was increased. Analysis by transmission electron microscopy of an S. coelicolor strain overexpressing ssgA showed that septum formation had strongly increased in comparison to wild-type S. coelicolor, showing that SsgA clearly influences Streptomyces cell division. The morphology of the hyphae was affected such that irregular septa were produced with a significantly wider diameter, thereby forming spore-like compartments. This suggests that ssgA can induce a process similar to submerged sporulation in Streptomyces strains that otherwise fail to do so. A working model is proposed for the regulation of septum formation and of submerged sporulation.

Translational regulation by modifications of the elongation factor Tu.

EF-Tu from E. coli, one of the superfamily of GTPase switch proteins, plays a central role in the fast and accurate delivery of aminoacyl-tRNAs to the translating ribosome. An overview is given about the regulatory effects of methylation, phosphorylation and phage-induced cleavage of EF-Tu on its function. During exponential growth, EF-Tu becomes monomethylated at Lys56 which is converted to Me2Lys upon entering the stationary phase. Lys56 is in the GTPase switch-1 region (residues 49-62), a strongly conserved site involved in interactions with the nucleotide and the 5' end of tRNA. Methylation was found to attenuate GTP hydrolysis and may thus enhance translational accuracy. In vivo 5-10% of EF-Tu is phosphorylated at Thr382 by a ribosome-associated kinase. In EF-Tu-GTP, Thr382 in domain 3 has a strategic position in the interface with domain 1; it is hydrogen-bonded to Glu117 that takes part in the switch-2 mechanism, and is close to the T-stem binding site of the tRNA, in a region known for many kirromycin-resistance mutations. Phosphorylation is enhanced by EF-Ts, but inhibited by kirromycin. In reverse, phosphorylated EF-Tu has an increased affinity for EF-Ts, does not bind kirromycin and can no longer bind aminoacyi tRNA. The in vivo role of this reversible modification is still a matter of speculation. T4 infection of E. coli may trigger a phase-exclusion mechanism by activation of Lit, a host-encoded proteinase. As a result, EF-Tu is cleaved site-specifically between Gly59-Ile60 in the switch-1 region. Translation was found to drop beyond a minimum level. Interestingly, the identical sequence in the related EF-G appeared to remain fully intact. Although the Lit cleavage-mechanism may eventually lead to programmed cell death, the very efficient prevention of phage multiplication may be caused by a novel mechanism of in cis inhibition of late T4 mRNA translation.

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.

Either of the chromosomal tuf genes of E. coli K-12 can be deleted without loss of cell viability.

A method of lambda-mediated gene replacement was used to disrupt tufA or tufB on the chromosome of the E. coli K-12 strain MG1655. Both tuf genes, which are almost identical but map in different chromosomal contexts, encode the essential peptide chain elongation factor EF-Tu, one of the most abundant cytoplasmic proteins. Southern analysis confirmed replacement of the chromosomal tufA or tufB gene by a chloramphenicol resistance marker, demonstrating that both tuf genes are individually dispensable for growth. Under conditions of rapid growth, deletion of tufB had no significant effect on growth rate, but deletion of tufA resulted in a 35% increase in generation time. In minimal medium we observed no negative effects of tufA deletion on growth rate. Strains with a single tuf gene are useful for the expression of mutant forms of EF-Tu as the sole species in cells; this was demonstrated by introducing the hybrid tufAhis gene, encoding EF-TuA extended with a C-terminal (His)6 tag, into the chromosome of a strain lacking tufB.

Structure and expression of elongation factor Tu from Bacillus stearothermophilus.

The tuf gene coding for elongation factor Tu (EF-Tu) of Bacillus stearothermophilus was cloned and sequenced. This gene maps in the same context as the tufA gene of Escherichia coli str operon. Northern-blot analysis and primer extension experiments revealed that the transcription of the tuf gene is driven from two promoter regions. One of these is responsible for producing a 4.9-kb transcript containing all the genes of B. stearothermophilus str operon and the other, identified adjacent to the stop codon of the fus gene and designated tufp, for producing a 1.3-kb transcript of the tuf gene only. In contrast to the situation in E. coli, the ratio between the transcription products was found to be about 10:1 in favour of the tuf gene transcript. This high transcription activity from the tufp promoter might be accounted for by the presence of an extremely A+T-rich block consisting of 29 nucleotides which immediately precedes the consensus -35 region of the promoter. A very similar tuf gene transcription strategy and the same tufp promoter organization with the identical A/T block were found in Bacillus subtilis. The tuf gene specifies a protein of 395 amino acid residues with a molecular mass of 43,290 Da, including the N-terminal methionine. A computer-generated three-dimensional homology model shows that all the structural elements essential for binding guanine nucleotides and aminoacyl-tRNA are conserved. The presence of serine at position 376 and a low affinity for kirromycin determined by zone-interference gel electrophoresis (Kd approximately 8 microM) and by polyacrylamide gel electrophoresis under non-denaturing conditions are in agreement with the reported resistance of this EF-Tu to the antibiotic. The replacement of the highly conserved Leu211 by Met was identified as a possible cause of pulvomycin resistance.

Specific peptide-activated proteolytic cleavage of Escherichia coli elongation factor Tu.

Phage exclusion is a form of programmed cell death in prokaryotes in which death is triggered by infection with phage, a seemingly altruistic response that limits multiplication of the phage and its spread through the population. One of the best-characterized examples of phage exclusion is the exclusion of T-even phages such as T4 by the e14-encoded Lit protein in many Escherichia coli K-12 strains. In this exclusion system, transcription and translation of a short region of the major head coat protein gene late in phage infection activates proteolysis of translation elongation factor Tu (EF-Tu), blocking translation and multiplication of the phage. The cleavage occurs between Gly-59 and Ile-60 in the nucleotide-binding domain. In the present work, we show that a 29-residue synthetic peptide spanning the activating region of the major head coat protein can activate the cleavage of GDP-bound EF-Tu in a purified system containing only purified EF-Tu and purified Lit protein. Lit behaves as a bona fide enzyme in this system, cleaving EF-Tu to completion when present at substoichiometric amounts. Two mutant peptides with amino acid changes that reduce the activation of cleavage of EF-Tu in vivo were also greatly reduced in their ability to activate EF-Tu cleavage in vitro but were still able to activate cleavage at a high concentration. Elongation factor G, which has the same sequence at the cleavage site and a nucleotide-binding domain similar to EF-Tu, was not cleaved by this system, and neither was heat-inactivated EF-Tu, suggesting that the structural context of the cleavage site may be important for specificity. This system apparently represents an activation mechanism for proteolysis that targets one of nature's most evolutionarily conserved proteins for site-specific cleavage.

Growth phase-dependent transcription of the Streptomyces ramocissimus tuf1 gene occurs from two promoters.

The str operon of Streptomyces ramocissimus contains the genes for ribosomal proteins S12 (rpsL) and S7 (rpsG) and for the polypeptide chain elongation factors G (EF-G) (fus) and Tu (EF-Tu) (tuf). This kirromycin producer contains three tuf or tuf-like genes; tuf1 encodes the regular EF-Tu and is located immediately downstream of fus. In vivo and in vitro transcription analysis revealed a transcription start site directly upstream of S. ramocissimus tuf1, in addition to the operon promoter rpsLp. Transcription from these promoters appeared to be growth phase dependent, diminishing drastically upon entry into stationary phase and at the onset of production of the EF-Tu-targeted antibiotic kirromycin. In surface-grown cultures, a second round of tuf1 transcription, coinciding with aerial mycelium formation and kirromycin production, was observed. The tuf1-specific promoter (tuf1p) was located in the intercistronic region between fus and tuf1 by high-resolution S1 mapping, in vitro transcription, and in vivo promoter probing. During logarithmic growth, the tuf1p and rpsLp transcripts are present at comparable levels. In contrast to Escherichia coli, which has two almost identical tuf genes, the gram-positive S. ramocissimus contains only tuf1 for its regular EF-Tu. High levels of EF-Tu may therefore be achieved by the compensatory activity of tuf1p.

Elongation factor Tu1 of the antibiotic GE2270A producer Planobispora rosea has an unexpected resistance profile against EF-Tu targeted antibiotics.

Sensitivity of EF-Tu1 of the GE2270A producer Planobispora rosea towards GE2270A, pulvomycin and kirromycin was determined by band-shift assays for EF-Tu1-antibiotic complex formation and by in vitro translation experiments. EF-Tu1 of P. rosea appeared to be not only totally resistant to GE2270A, but also ten times more resistant to kirromycin than EF-Tu1 of Streptomyces coelicolor. In contrast, P. rosea EF-Tu1 was found to be not resistant to pulvomycin, an antibiotic that just like GE2270A blocks EF-Tu x GTP x aminoacyl-tRNA complex formation. Previous in vivo and in vitro experiments with mixed populations of antibiotic resistant and sensitive EF-Tu species had shown that sensitivity to kirromycin and pulvomycin is dominant over resistance. In the case of GE2270A we observed, however, that sensitivity is recessive to resistance, which again points to a different action mechanism than in the case of pulvomycin. Besides the tuf1 gene encoding the regular elongation factor EF-Tu1 a gene similar to S. coelicolor tuf3 for a specialized EF-Tu was located in the P. rosea genome. The tuf1 gene was isolated and sequenced. The amino acid sequence of EF-Tul of P. rosea not only exhibits an unusual Tyr160 substitution (comparable to those described for kirromycin-resistant EF-Tus), but also shows significant changes of conserved amino acids in domain 2 that may be responsible for GE2270A resistance (the latter do not resemble those leading to pulvomycin resistance). P. rosea EF-Tu1 thus is a first example of a bacterial EF-Tu with resistance against two divergently acting antibiotics.

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.

Translational activities of EF-Tu [G222D] which cannot be reconciled with the classical scheme of the polypeptide chain elongation cycle.

We have developed a cell-free system of E. coli that enables us to study the in vitro translation of natural mRNA mediated by wild-type or mutant EF-Tu. Various mutant EF-Tu species have been analyzed, one of which, EF-Tu [G222D], appeared to be virtually unable to mediate the translation of natural mRNA. Since this mutant factor is able to participate in translation in vivo by suppressing nonsense and frameshift mutations in cooperation with EF-Tu [A375T], a revision of the generally accepted scheme of the elongation cycle has been proposed (Bosch, L., Vijgenboom, E., & Zeef, L.A.H., 1996, Biochemistry 36).

A growth-defective kirromycin-resistant EF-Tu Escherichia coli mutant and a spontaneously evolved suppression of the defect.

This study has investigated the cause of a growth-defect phenotype of a mutation in the elongation factor EF-Tu from Escherichia coli. An M13-based genetic retrieval system reported by Zeef and Bosch [Mol. Gen. Genet. 238 (1993) 252-260] was used to segregate and identify an extremely growth-defective kirromycin-resistant (KrR) tufA mutation, encoding Gln124-->Lys (Q124K), from a KrR parent strain. This original strain also contained mutations, 124com1 and 124com2, that appear to have evolved to suppress the Q124K tufA mutation. In this communication we present these M13-based genetic experiments together with additional genetic and protein characterization experiments to clarify the basis of this complementation. The data indicate that the serious growth defect of Q124K originates from a defective GTP/GDP interaction. The GTP/GDP binding and GTP hydrolysis characteristics of ET-Tu Q124K were different from wild-type EF-Tu and especially of another KrR EF-Tu mutant A375T. In line with this, 124com1 specifically complemented EF-Tu Q124K, whereas the growth defects of strains containing EF-Tu mutated at aa 375 were aggravated. We also show that strains containing the segregated tufA Q124K mutation formed filaments.