2D label-free imaging of resonant grating biochips in ultraviolet.

2D images of label-free biochips exploiting resonant waveguide grating (RWG) are presented. They indicate sensitivities on the order of 1 pg/mm2 for proteins in air, and hence 10 pg/mm2 in water can be safely expected. A 320x256 pixels Aluminum-Gallium-Nitride-based sensor array is used, with an intrinsic narrow spectral window centered at 280 nm. The additional role of characteristic biological layer absorption at this wavelength is calculated, and regimes revealing its impact are discussed. Experimentally, the resonance of a chip coated with protein is revealed and the sensitivity evaluated through angular spectroscopy and imaging. In addition to a sensitivity similar to surface plasmon resonance (SPR), the RWGs resonance can be flexibly tailored to gain spatial, biochemical, or spectral sensitivity.

Solution structure of the anticodon-binding domain of Escherichia coli lysyl-tRNA synthetase and studies of its interaction with tRNA(Lys).

A protein domain corresponding to residues 31 to 149 of the E. coli Lysyl-tRNA synthetase species corresponding to the lysS gene was expressed and 15N-labelled. 1H and 15N NMR resonance assignments for this domain were obtained by two-dimensional and three-dimensional homonuclear and heteronuclear spectroscopy. Using distance geometry and simulated annealing, a three-dimensional structure could be calculated using 701 NOE and 86 dihedral angle restraints. It is composed of a five-stranded antiparallel beta-barrel capped by three alpha-helices at its ends. This structure closely resembles that of the N-terminal domain of the other E. coli lysyl-tRNA synthetase species expressed from the lysU gene and is highly homologous to the fold observed for the corresponding region of aspartyl-tRNA synthetase. It is shown that the isolated N-terminal fragment of lysyl-tRNA synthetase can interact with tRNA(Lys) as well as with poly (U), which mimics the anticodon sequence. Amino acid residues involved in these interactions were identified and, in the case of poly-U, a number of specific protein-RNA contacts were characterized. Specific recognition of tRNA(Lys) involves a cluster of four structurally well-defined aromatic residues, anchored on the beta-strands, and basic residues located on the surrounding loops. This organization is reminiscent of other RNA binding proteins, such as the U1A small nuclear ribonucleoprotein.

Design and characterization of Escherichia coli mutants devoid of Ap4N-hydrolase activity.

Escherichia coli strains with abnormally high concentrations of bis(5'-nucleosidyl)-tetraphosphates (Ap4N) were constructed by disrupting the apaH gene that encodes Ap4N-hydrolase. Variation deletions and insertions were also introduced in apaG and ksgA, two other cistrons of the ksgA apaGH operon. In all strains studied, a correlation was found between the residual Ap4N-hydrolase activity and the intracellular Ap4N concentration. In cells that do not express apaH at all, the Ap4N concentration was about 100-fold higher than in the parental strain. Such a high Ap4N level did not modify the bacterial growth rate in rich or minimal medium. However, while, as expected, the ksgA- and apaG- ksgA- strains stopped growing in the presence of this antibiotic at 600 micrograms/ml. The were not sensitive to kasugamycin, the apaH- apaG- ksgA- strain filamented and stopped growing in the presence of this antibiotic at 600 micrograms/ml. The growth inhibition was abolished upon complementation with a plasmid carrying an intact apaH gene. Trans addition of extra copies of the heat-shock gene dnaK also prevented the kasugamycin-induced filamentation of apaH- apaG- ksgA- strains. This result is discussed in relation to the possible involvement of Ap4N in cellular adaptation following a stress.

tRNA anticodon recognition and specification within subclass IIb aminoacyl-tRNA synthetases.

Subclass IIb aminoacyl-tRNA synthetases (Asn-, Asp- and LysRS) recognize the anticodon triplet of their cognate tRNA (GUU, GUC and UUU, respectively) through an OB-folded N-terminal extension. In the present study, the specificity of constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli was analyzed by cross-mutagenesis of the tRNA(Lys) anticodon, on the one hand, and of the amino acid residues composing the anticodon binding site on the other. From this analysis, a tentative model is deduced for both the recognition of the cognate anticodon and the rejection of non-cognate anticodons. In this model, the enzyme offers a rigid scaffold of amino acid residues along the beta-strands of the OB-fold for tRNA binding. Phe85 and Gln96 play a critical role in this spatial organization. This scaffold can recognize directly U35 at the center of the anticodon. Specification of the correct enzyme:tRNA complex is further achieved through the accommodation of U34 and U36. The binding of these bases triggers the conformationnal change of a flexible seven-residue loop between strands 4 and 5 of the OB-fold (L45). Additional free energy of binding is recovered from the resulting network of cooperative interactions. Such a mechanism would not depend on the modifications of the anticodon loop of tRNA(Lys) (mnm5s2U34 and t6A37). In the model, exclusion by the synthetase of non-cognate anticodons can be accounted for by a hindrance to the positioning of the L45 loop. In addition, Glu135 would repulse a cytosine base at position 35. Sequence comparisons show that the composition and length of the L45 loop are markedly conserved in each of the families composing subclass IIb aminoacyl-tRNA synthetases. The possible role of the loop is discussed for each case, including that of archaebacterial aspartyl-tRNA synthetases.

Structure-function relationship of the Lrp-binding region upstream of lysU in Escherichia coli.

In Escherichia coli, one of the two genes encoding lysyl-tRNA synthetase, lysU, belongs to the regulon controlled by the leucine-responsive regulatory protein (Lrp). To map the site of Lrp action, mutants escaping regulation in rich medium were generated through random mutagenesis of the lysU promoter region. The mutations showed parallel effects on the strength of Lrp-DNA association, as measured in vitro by gel retardation experiments, and on the degree of repression of lysU expression by Lrp in vivo. In addition, DNase I and hydroxyl radical footprinting experiments indicated that several Lrp molecules bind to a DNA region of over 110 bp in a highly cooperative manner. This region, which encompasses the -35 box of the lysU promoter, was the target of all the mutations affecting the strength of the Lrp-DNA association. These mutations are frequently located in short A + T-rich runs distributed along the Lrp binding region with a periodicity of one helix turn. Because we could find such a regular alternance of A + T runs upstream of several other Lrp-regulated genes, we suggest that this pattern is one feature indicative of the binding of Lrp.

Crystallization and preliminary diffraction studies of Escherichia coli lysyl-tRNA synthetase (LysU).

Crystals of Escherichia coli lysyl-tRNA synthetase (lysU gene product) have been obtained by vapour diffusion techniques. Three different crystal forms could be grown under similar conditions. The crystals that have been chosen for the structure determination belong to space group C222(1) with cell dimensions a = 144.3 A, b = 257.8 A, c = 182.1 A and contain three monomers in the asymmetric unit. They diffract to at least 2.1 A resolution, but are very sensitive to radiation damage.

Evidence for a new Escherichia coli protein resembling a lysyl-tRNA synthetase.

The amino acid sequence deduced from the nucleotide sequence of an open reading frame adjacent to the frdA gene of Escherichia coli shows 30.5% identity with the C terminus of Escherichia coli lysyl-tRNA synthetases. The three motifs characteristic of aminoacyl-tRNA synthetases of class 2 are recognizable within this sequence.

Escherichia coli leucine-responsive regulatory protein (Lrp) controls lysyl-tRNA synthetase expression.

Using random Tn10 insertion mutagenesis, we isolated an Escherichia coli mutant strain affected in the regulation of lysU, the gene encoding the inducible form of lysyl-tRNA synthetase. The transposon giving rise to the altered expression of lysU was found inserted within lrp. The latter gene codes for the leucine-responsive regulatory protein (Lrp) which mediates a global response of the bacterium to leucine. An involvement of Lrp in the regulation of lysU was searched for by using a lysU-lacZ operon fusion. The following conclusions were reached: (i) inactivation of lrp causes an increased activity of the lysU promoter, whatever the growth conditions assayed, (ii) insertion of a wild-type lrp gene into a multi-copy plasmid significantly reduces lysU expression, and (iii) sensitivity of the lysU promoter to the presence of leucine in the growth medium is abolished in the lrp context.

Sequence, overproduction and crystallization of aspartyl-tRNA synthetase from Thermus thermophilus. Implications for the structure of prokaryotic aspartyl-tRNA synthetases.

The genes of aspartyl-tRNA synthetase (AspRS) from two Thermus thermophilus strain VK-1 and HB8, have been cloned and sequenced. Their nucleotidic sequences code for the same protein which displays the three characteristic motifs of class II aminoacyl-tRNA synthetases. This enzyme shows 50% identity with Escherichia coli AspRS, over the totality of the chain (580 amino acids). A comparison with the eukaryotic yeast cytoplasmic AspRS indicates the presence in the prokaryotic AspRS of an extra domain between motifs 2 and 3 much larger than in the eukaryotic ones. When its gene is under the control of the tac promoter of the expression vector pKK223-3, the protein is efficiently overexpressed as a thermostable protein in E. coli. It can be further purified to homogeneity using a heat treatment followed by a single anion exchange chromatography. Single crystals of the pure protein, diffracting at least to 2.2 A resolution (space group P2(1)2(1)2(1), a = 61.4 A, b = 156.1 A, c = 177.3 A) are routinely obtained. The same crystals have previously been described as crystals of threonyl-tRNA synthetase [1].

Aminoacyl-tRNA synthetases: a new image for a classical family.

The aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes well known for their role in protein synthesis. More recent investigations have discovered that this classic family of enzymes is actually capable of a broad repertoire of functions which not only impact protein synthesis, but extend to a number of other critical cellular activities. Specific aaRSs play roles in cellular fidelity, tRNA processing, RNA splicing, RNA trafficking, apoptosis, transcriptional and translational regulation. A recent EMBO workshop entitled 'Structure and Function of Aminoacyl-tRNA Synthetases' (Mittelwihr, France, October 10-15, 1998), highlighted the diversity of the aaRSs' role within the cell. These novel activities as well as significant advances in delineating mechanisms of substrate specificity and the aminoacylation reaction affirm the family of aaRSs as pharmaceutical targets.

Internal motions of transfer RNA: a study of exchanging protons by magnetic resonance.

Proton exchange is a probe of macromolecular structure and kinetics. Its value is enhanced when the exchanging protons can be identified by nmr. After dilution of tRNA-H2O samples in D2O, slowly exchanging imino protons are observed, with exchange times ranging from minutes to days. In many cases they originate from the dihydro-uracil region. Most slow exchangers are sensitive to buffer catalysis. Extrapolation to infinite buffer concentration yields the life-time of the closed form, in a two-state model of each base-pair. As predicted by the model, the lifetime obtained by extrapolation is independent of the buffer. Typical lifetimes are 14 minutes for CG11 of yeast tRNAPhe at 17 degrees C, or 5 minutes for U8-A14 of yeast tRNA(Asp) at 20 degrees C, without magnesium. For most slow exchangers, magnesium increases the lifetime of the closed form, but moderately, by factors never more than five. The exchange rates of other, fast-exchanging, imino protons, as determined by line-broadening, are found to depend on buffer concentration. Base-pair lifetimes are determined as above. For instance UA6 of yeast tRNA(Phe) has a lifetime of 14 ms at 17 degrees C. Base-pairs 4 and 6 have shorter lifetimes than the rest of the acceptor stem. Imidazole is a good catalyst for proton exchange of both the long-and the short-lived base-pairs, whereas phosphate is not. Tris is efficient except for cases where, possibly, access is impeded by its size; magnesium reduces the efficiency of catalysis by tris buffer. From the variation of exchange time vs buffer concentration, one determines the buffer concentration for which the exchange rate from the open state is equal to the closing rate. Remarquably, this concentration takes comparable values for most base-pairs, whether short-lived or long-lived. Buffer effects have also been observed in poly(rA).poly(rU), for which we derive a lifetime of 2.5 ms at 27 degrees C, and in other polynucleotides. Some of the exchange times identified in the literature as base-pair lifetimes may instead reflect incomplete catalysis.

The role of zinc in 5',5'-diadenosine tetraphosphate production by aminoacyl-transfer RNA synthetases.

Aminoacyl-tRNA synthetases are capable of converting 5'-ATP into 5',5'-diadenosine tetraphosphate. The reaction reflects the reversal of enzyme-bound aminoacyl-adenylate by ATP instead of PPi. In the case of a few prokaryotic as well as eukaryotic aminoacyl-tRNA synthetases, the initial rate of diadenosine tetraphosphate synthesis can be greatly enhanced upon adding small amounts of zinc. This observation enables us to establish a relationship between diadenosine tetraphosphate, a nucleotide possibly involved in controlling cell proliferation, and a metallic cofactor, which is believed to play a role in tumour growth.

A single substitution in the motif 1 of Escherichia coli lysyl-tRNA synthetase induces cooperativity toward amino acid binding.

The constitutive lysyl-tRNA synthetase (LysRS) of the Escherichia coli strain OEL134 differs from the wild-type enzyme by the single substitution of threonine 208 with methionine. In vitro study of the isotopic [32P]PPi-ATP exchange reaction catalyzed by purified T208M LysRS revealed specific features that are not observed with the wild-type LysRS: (i) The steady state of the reaction was reached after a approximately 1-min lag when the addition of the enzyme was used to initiate the reaction. This lag disappeared upon preincubation of the enzyme with lysine and ATP. (ii) The variation of the steady state rate as a function of the lysine concentration in the assay was sigmoidal (Hill coefficient of 1.65), suggesting cooperativity of lysine binding to this dimeric enzyme. The allosteric behavior of the mutant enzyme was further established by showing that, at low concentrations of lysine, low amounts of cadaverine stimulated T208M LysRS activity. T208A LysRS, in which threonine 208 had been changed into alanine by site-directed mutagenesis, displayed the same properties as T208M LysRS. Remarkably, Thr 208 makes part of the first signature motif of class II aminoacyl-tRNA synthetases, a motif likely to be involved in the dimerization of the enzyme subunits. Therefore, the behavior of the Thr 208 mutants of LysRS supports the idea that the dimerization of class II aminoacyl-tRNA synthetases is important for an efficient structuration of their active site.

Function of the extra 5'-phosphate carried by histidine tRNA.

Among elongator tRNAs, tRNA specific for histidine has the peculiarity to possess one extra nucleotide at position -1. This nucleotide is believed to be responsible for recognition by histidyl-tRNA synthetase. Here, we show that, in fact, it is the phosphate 5' to the extra nucleotide which mainly supports the efficiency of the tRNA aminoacylation reaction catalyzed by Escherichia coli histidyl-tRNA synthetase. In the case of the reaction of E. coli peptidyl-tRNA hydrolase, this atypical phosphate is dispensable. Instead, peptidyl-tRNA hydrolase recognizes the phosphate of the phosphodiester bond between residues -1 and +1 of tRNA(His). Recognition of the +1 phosphate of tRNA(His) by peptidyl-tRNA hydrolase resembles, therefore, that of the 5'-terminal phosphate of other elongator tRNAs.

D-tyrosyl-tRNA(Tyr) metabolism in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae YDL219w (DTD1) gene, which codes for an amino acid sequence sharing 34% identity with the Escherichia coli D-Tyr-tRNA(Tyr) deacylase, was cloned, and its product was functionally characterized. Overexpression in the yeast of the DTD1 gene from a multicopy plasmid increased D-Tyr-tRNA(Tyr) deacylase activity in crude extracts by two orders of magnitude. Upon disruption of the chromosomal gene, deacylase activity was decreased by more than 90%, and the sensitivity to D-tyrosine of the growth of S. cerevisiae was exacerbated. The toxicity of D-tyrosine was also enhanced under conditions of nitrogen starvation, which stimulate the uptake of D-amino acids. In relation with these behaviors, the capacity of purified S. cerevisiae tyrosyl-tRNA synthetase to produce D-Tyr-tRNA(Tyr) could be shown. Finally, the phylogenetic distribution of genes homologous to DTD1 was examined in connection with L-tyrosine prototrophy or auxotrophy. In the auxotrophs, DTD1-like genes are systematically absent. In the prototrophs, the putative occurrence of a deacylase is variable. It possibly depends on the L-tyrosine anabolic pathway adopted by the cell.

Direct random mutagenesis of gene-sized DNA fragments using polymerase chain reaction.

The polymerase chain reaction (PCR) can be used to amplify a DNA fragment with the concomitant creation of numerous mutations provided that one dNTP substrate is in excess over the three others. Advantage was taken of this behavior to systematically mutagenize a 291-bp-long DNA fragment and to define the rules relating the frequencies of each possible bp substitution to the set of the dNTP concentrations in the PCR experiment. Sets of parameters governing the rules were determined under various mutagenic conditions including the addition of MnCl2. Finally, validity of the rules was assessed in several mutagenesis experiments showing that a wide range of substitution frequencies including AT-->GC and GC-->AT transitions as well as AT-->TA transversions can be obtained at will.

Metabolism of D-aminoacyl-tRNAs in Escherichia coli and Saccharomyces cerevisiae cells.

In Escherichia coli, tyrosyl-tRNA synthetase is known to esterify tRNA(Tyr) with tyrosine. Resulting d-Tyr-tRNA(Tyr) can be hydrolyzed by a d-Tyr-tRNA(Tyr) deacylase. By monitoring E. coli growth in liquid medium, we systematically searched for other d-amino acids, the toxicity of which might be exacerbated by the inactivation of the gene encoding d-Tyr-tRNA(Tyr) deacylase. In addition to the already documented case of d-tyrosine, positive responses were obtained with d-tryptophan, d-aspartate, d-serine, and d-glutamine. In agreement with this observation, production of d-Asp-tRNA(Asp) and d-Trp-tRNA(Trp) by aspartyl-tRNA synthetase and tryptophanyl-tRNA synthetase, respectively, was established in vitro. Furthermore, the two d-aminoacylated tRNAs behaved as substrates of purified E. coli d-Tyr-tRNA(Tyr) deacylase. These results indicate that an unexpected high number of d-amino acids can impair the bacterium growth through the accumulation of d-aminoacyl-tRNA molecules and that d-Tyr-tRNA(Tyr) deacylase has a specificity broad enough to recycle any of these molecules. The same strategy of screening was applied using Saccharomyces cerevisiae, the tyrosyl-tRNA synthetase of which also produces d-Tyr-tRNA(Tyr), and which, like E. coli, possesses a d-Tyr-tRNA(Tyr) deacylase activity. In this case, inhibition of growth by the various 19 d-amino acids was followed on solid medium. Two isogenic strains containing or not the deacylase were compared. Toxic effects of d-tyrosine and d-leucine were reinforced upon deprivation of the deacylase. This observation suggests that, in yeast, at least two d-amino acids succeed in being transferred onto tRNAs and that, like in E. coli, the resulting two d-aminoacyl-tRNAs are substrates of a same d-aminoacyl-tRNA deacylase.

Role of D-cysteine desulfhydrase in the adaptation of Escherichia coli to D-cysteine.

D-cysteine, a powerful inhibitor of Escherichia coli growth, is decomposed in vitro into pyruvate, H2S, and NH3 by D-cysteine desulfhydrase. To assess the role of this reaction in the adaptation of the bacterium to growth on D-cysteine, the gene of the desulfhydrase was cloned. It corresponds to the open reading frame yedO at 43.03 min on the genetic map of E. coli. The amino acid sequence deduced from this gene is homologous to those of several 1-aminocyclopropane-carboxylate deaminases. However, the E. coli desulfhydrase does not use 1-aminocyclopropane-1-carboxylate as substrate. Various mutants in which the yedO gene was inactivated or overexpressed were constructed. They exhibited hypersensitivity or resistance, respectively, to the presence of d-cysteine in the culture medium. Growth protection against D-cysteine in minimal medium was conferred by the simultaneous addition of isoleucine, leucine, and valine. In agreement with this behavior, D-cysteine inhibited the activity of threonine deaminase, a key enzyme of the isoleucine, leucine, and valine pathway. Finally, in the presence of the intact yedO gene, E. coli growth was improved by addition of D-cysteine as the sole sulfur source. In agreement with a role of the desulfhydrase in sulfur metabolism, yedO expression was induced under conditions of sulfate limitation.