G-Domain Dimerization Orchestrates the tRNA Wobble Modification Reaction in the MnmE/GidA Complex

MnmE and GidA are involved in the modification of wobble uridine to carboxymethylaminomethyl uridine in certain tRNAs. Malfunctioning of the human orthologs has been implicated in mitochondrial diseases. MnmE is a conserved G protein activated by dimerization. Here, we show that complex formation between MnmE and GidA involves large conformational changes that induce G-domain dimerization of MmnE and that GidA co-stimulates GTP hydrolysis on MnmE. Starting from a structural model of the complex, we identify interface mutations disrupting complex formation or communication. Although GidA does not directly contact the G-domains, conformational changes in MnmE, induced by G-domain dimerization in the triphosphate state, regulate the affinity for GidA. We developed a tRNA modification assay and demonstrate for the first time in vitro that the MnmE/GidA complex catalyzes incorporation of glycine into tRNA. An intact MnmE/GidA complex rather than their sequential action is crucial for in vitro modification. Since only GTP, but not GDP or non-hydrolyzable GTP analogs, drives the MnmE/GidA-catalyzed modification reaction, we conclude that GTP hydrolysis is essential for activity. We finally show that an active GTPase, an intact MnmE/GidA communication, and dimerization of G-domains are necessary for in vivo functioning since mutations disrupting either result in a respiratory deficient phenotype in yeast.     

Effects of mutagenesis in the switch I region and conserved arginines of Escherichia coli MnmE protein, a GTPase involved in tRNA modification

MnmE is an evolutionarily conserved, three domain GTPase involved in tRNA modification. In contrast to Ras proteins, MnmE exhibits a high intrinsic GTPase activity and requires GTP hydrolysis to be functionally active. Its G domain conserves the GTPase activity of the full protein, and thus, it should contain the catalytic residues responsible for this activity. In this work, mutational analysis of all conserved arginine residues of the MnmE G-domain indicates that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis. In addition, we show that residues in the G2 motif (249GTTRD253), which resides in the switch I region, are not important for GTP binding but play some role in stabilizing the transition state, specially Gly249 and Thr251. On the other hand, G2 mutations leading to a minor loss of the GTPase activity result in a non-functional MnmE protein. This indicates that GTP hydrolysis is a required but non-sufficient condition so that MnmE can mediate modification of tRNA. The conformational change of the switch I region associated with GTP hydrolysis seems to be crucial for the function of MnmE, and the invariant threonine (Thr251) of the G2 motif would be essential for such a change, because it cannot be substituted by serine. MnmE defects result in impaired growth, a condition that is exacerbated when defects in other genes involved in the decoding process are simultaneously present. This behavior is reminiscent to that found in yeast and stresses the importance of tRNA modification for gene expression.     

Activation of protease-constitutive recA proteins of Escherichia coli by rRNA and tRNA.

The RecA proteins of the unusually strong protease-constitutive mutants recA1202 and recA1211 can use RNA in addition to single-stranded DNA (ssDNA) as a cofactor in the cleavage of the LexA repressor in vitro. In the presence of rRNA or tRNA, the effectiveness of these proteins decreased in the order RecA1202 greater than RecA1211 much greater than RecA+, which is also the order of their in vivo constitutive protease activities. The effectiveness of rRNA was comparable to that of ssDNA in the cleavage of the LexA repressor by either mutant protease. Although all the common nucleoside triphosphates can act as positive effectors for LexA cleavage by the two mutant proteins in the presence of ssDNA (W. B. Wang, M. Sassanfar, I. Tessman, J. W. Roberts, and E. S. Tessman, J. Bacteriol. 170:4816-4822, 1988), only dATP, ATP, and ATP-gamma-S were effective in the presence of RNA. Our results explain more fully why certain recA mutants have high constitutive protease activities in vivo.     

The GTPase activity and C-terminal cysteine of the Escherichia coli MnmE protein are essential for its tRNA modifying function

The Escherichia coli MnmE protein is a three-domain protein that exhibits a very high intrinsic GTPase activity and low affinity for GTP and GDP. The middle GTPase domain, when isolated, conserves the high intrinsic GTPase activity of the entire protein, and the C-terminal domain contains the only cysteine residue present in the molecule. MnmE is an evolutionarily conserved protein that, in E. coli, has been shown to control the modification of the uridine at the wobble position of certain tRNAs. Here we examine the biochemical and functional consequences of altering amino acid residues within conserved motifs of the GTPase and C-terminal domains of MnmE. Our results indicate that both domains are essential for the MnmE tRNA modifying function, which requires effective hydrolysis of GTP. Thus, it is shown for the first time that a confirmed defect in the GTP hydrolase activity of MnmE results in the lack of its tRNA modifying function. Moreover, the mutational analysis of the GTPase domain indicates that MnmE is closer to classical GTPases than to GTP-specific metabolic enzymes. Therefore, we propose that MnmE uses a conformational change associated with GTP hydrolysis to promote the tRNA modification reaction, in which the C-terminal Cys may function as a catalytic residue. We demonstrate that point mutations abolishing the tRNA modifying function of MnmE confer synthetic lethality, which stresses the importance of this function in the mRNA decoding process.     

The ribosome and YidC. New insights into the biogenesis of Escherichia coli inner membrane proteins

In the bacterium Escherichia coli, inner membrane proteins (IMPs) are generally targeted through the signal recognition particle pathway to the Sec translocon, which is capable of both linear transport into the periplasm and lateral transport into the lipid bilayer. Lateral transport seems to be assisted by the IMP YidC. In this article, we discuss recent observations that point to a key role for the ribosome in IMP targeting and to the diverse roles of YidC in IMP assembly.     

Queuosine modification in tRNA and expression of the nitrate reductase in Escherichia coli.

In eubacteria the modified nucleoside queuosine is present in tRNAAsn, tRNAAsp, tRNAHis and tRNATyr. A precursor of queuine, pre-queuine, is synthesized from GTP, inserted into the first position of the anticodon of the corresponding tRNAs by a specific tRNA-guanine transglycosylase and further modified to queuosine. Isogenic pairs of Escherichia coli, containing or lacking the tRNA-transglycosylase (JE 7335, tgt+ lacZ+ and JE 7337, tgt- lacZ+; JE 7334, tgt+ lacZ- and JE 7336, tgt- lacZ-), have been employed to study the function of queuosine in tRNA. Compared with the tgt+ strain (JE 7335), the tgt- mutant (JE 7337) grown under anaerobic conditions, is defective with respect to the nitrate respiration system, in which electrons are transported from D(-)-lactate via quinone and cytochrome bNO3-(556) to nitrate. Low temperature cytochrome spectra of the anaerobically grown tgt- mutant show a lowered amount of type b cytochromes involving the spectrum of cytochrome bNO3-(556). In the case of the anaerobically grown tgt- mutant three proteins are missing in the protein pattern of cytoplasmic membranes. Their mol. wts. correspond to those of the subunits of the nitrate reductase complex. In contrast to the tgt+ strains (JE 7334, JE 7335) both tgt- mutants (JE 7336, JE 7337) cannot grow on lactate under anaerobic conditions with nitrate offered as electron acceptor and NO3- is not reduced to NO2-. A possible link between Q-modification of tRNAs, the synthesis of proteins of the nitrate reductase complex and the synthesis of menaquinone or ubiquinone is discussed.     

High-molecular-weight forms of aminoacyl-tRNA synthetases and tRNA modification enzymes in Escherichia coli.

The presence of high-molecular-weight complexes of aminoacyl-tRNA synthetases in Escherichia coli has been reported (C. L. Harris, J. Bacteriol. 169:2718-2723, 1987). In the current study, Bio-Gel A-5M gel chromatography of 105,000 x g supernatant preparations from E. coli Q13 indicated high molecular weights for both tRNA methylase (300,000) and tRNA sulfurtransferase (450,000). These tRNA modification enzymes did not appear to exist in the same multienzymic complex. On the other hand, 4-thiouridine sulfurtransferase eluted with aminoacyl-tRNA synthetase activity on Bio-Gel A-5M, and both of these activities were cosedimented after further centrifugation of cell supernatants at 160,000 x g for 18 h. Despite this evidence for association of the sulfurtransferase with the synthetase complex, isoleucyl-tRNA synthetase and tRNA sulfurtransferase were totally resolved from each other by DEAE-Sephacel chromatography. Subsequent gel chromatography showed little change in their elution positions on agarose. Hence, either nonspecific aggregation occurred here, or the modification enzymes studied are not members of the aminoacyl-tRNA synthetase complex in E. coli. These findings do suggest that some bacterial tRNA modification enzymes are present in multiprotein complexes of high molecular weight.     

Structural insights into the catalytic mechanism of Escherichia coli selenophosphate synthetase

Selenophosphate synthetase (SPS) catalyzes the synthesis of selenophosphate, the selenium donor for the biosynthesis of selenocysteine and 2-selenouridine residues in seleno-tRNA. Selenocysteine, known as the 21st amino acid, is then incorporated into proteins during translation to form selenoproteins which serve a variety of cellular processes. SPS activity is dependent on both Mg(2+) and K(+) and uses ATP, selenide, and water to catalyze the formation of AMP, orthophosphate, and selenophosphate. In this reaction, the gamma phosphate of ATP is transferred to the selenide to form selenophosphate, while ADP is hydrolyzed to form orthophosphate and AMP. Most of what is known about the function of SPS has derived from studies investigating Escherichia coli SPS (EcSPS) as a model system. Here we report the crystal structure of the C17S mutant of SPS from E. coli (EcSPS(C17S)) in apo form (without ATP bound). EcSPS(C17S) crystallizes as a homodimer, which was further characterized by analytical ultracentrifugation experiments. The glycine-rich N-terminal region (residues 1 through 47) was found in the open conformation and was mostly ordered in both structures, with a magnesium cofactor bound at the active site of each monomer involving conserved aspartate residues. Mutating these conserved residues (D51, D68, D91, and D227) along with N87, also found at the active site, to alanine completely abolished AMP production in our activity assays, highlighting their essential role for catalysis in EcSPS. Based on the structural and biochemical analysis of EcSPS reported here and using information obtained from similar studies done with SPS orthologs from Aquifex aeolicus and humans, we propose a catalytic mechanism for EcSPS-mediated selenophosphate synthesis.     

Topography of the Escherichia coli 30 S ribosome revealed by the modification of ribosomal proteins

Chemical and enzymatic iodination were used to determine the position of proteins within the 30 S ribosome of Escherichia coli. The relative degree of iodination was determined in intact 30 S ribosomes, particles unfolded with EDTA, ribonuclease digests of ribosomes and extracted proteins. These procedures permitted an evaluation of the influence of protein-RNA and protein-protein interactions, protein conformation and position, on the degree to which protein could be modified by radioactive iodine. From the data we conclude that all 30 S ribosomal proteins are accessible to the external milieu, and that none are buried within the three-dimensional structure of the particle.     

New insights into the ATP-dependent Clp protease: Escherichia coli and beyond

Proteolysis functions as a precise regulatory mechanism for a broad spectrum of cellular processes. Such control impacts not only on the stability of key metabolic enzymes but also on the effective removal of terminally damaged polypeptides. Much of this directed protein turnover is performed by proteases that require ATP and, of those in bacteria, the Clp protease from Escherichia coli is one of the best characterized to date. The Clp holoenzyme consists of two adjacent heptameric rings of the proteolytic subunit known as ClpP, which are flanked by a hexameric ring of a regulatory subunit from the Clp/Hsp100 chaperone family at one or both ends. The recently resolved three-dimensional structure of the E. coli ClpP protein has provided new insights into its interaction with the regulatory/chaperone subunits. In addition, an increasing number of studies over the last few years have recognized the added complexity and functional importance of ClpP proteins in other eubacteria and, in particular, in photosynthetic organisms ranging from cyanobacteria to higher plants. The goal of this review is to summarize these recent findings and to highlight those areas that remain unresolved.     

Secretion of recombinant proteins into the culture medium by Escherichia coli and Staphylococcus aureus

The ability of staphylococcal protein A (SPA) to bind to the Fc part of IgG has been used for the purification of a number of heterologous gene products as fusion proteins. Both the SPA promoter and signal sequence function in Escherichia coli, as well as in a number of Gram-positive bacteria, which facilitates comparisons of the expressed specific products in different hosts. The expression system developed for E. coli yields excretion of the fusion protein to the growth medium, which makes E. coli a competitive alternative to Gram-positive bacteria for the expression of secreted products. The human peptide hormones insulin-like growth factors (IGF) I and II were expressed using the protein A system in E. coli and Staphylococcus aureus. Despite a high degree of structural homology, large differences in the yields were observed in the two hosts. This underlines the importance of investigating different bacterial hosts for a particular protein product.     

Further temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins.

Summary Various alterations in ribosomal proteins were detected in forty-one mutants ofE. coli isolated as temperature-sensitive mutants. Out of these, six are new classes of mutants harboring mutations in proteins S3, L5, L7 (L12), L29, L30 and L33. One of them apparently lacks protein L7 of the large subunit. These mutants together with those reported previously (Isono et al., 1976) total one hundred and one ribosomal mutants in thirty different proteins.     

The effect of modification of tRNA nucleotide-37 on the tRNA interaction with the P- and A-site of the 70S ribosome Escherichia coli

A modified nucleotide on the 3'-side of the anticodon loop of tRNA is one of the most important structure element regulating codon-anticodone interaction on the ribosome owing to the stacking interaction with the stack of codon-anticodon bases. The presence and identity (pyrimidine, purine or modified purine) of this nucleotide has an essential influence on the energy of the stacking interaction on A- and P-sites of the ribosome. There is a significant influence of the 37-modification by itself on the P-site, whereas there is no such one on the A-site of the ribosome. Comparison of binding enthalpies of tRNA interactions on the P- or A-site of the ribosome with the binding enthalpies of the complex of two tRNAs with the complementary anticodones suggests that the ribosome by itself significantly endows in the thermodynamics of codon-anticodon complex formation. It happens by additional ribosomal interactions with the molecule of tRNA or indirectly by the stabilization of codon-anticodon conformation. In addition to the stacking, tRNA binding in the A and P sites is futher stabilized by the interactions involving some magnesium ions. The number of them involved in those interactions strongly depends on the nucleotide identity in the 37-position of tRNA anticodon loop.