Effect of variation of charged and uncharged tRNA(Trp) levels on ppGpp synthesis in Escherichia coli.

We introduced into a stringent Escherichia coli tryptophan auxotroph a plasmid bearing the tRNA(Trp) gene under the control of an inducible promoter. This allows us to manipulate the total concentration of tRNA(Trp) in the cell according to whether and when inducer is added to the culture. We also manipulated the concentration of Trp-tRNA(Trp) in vivo since the strain used bears a mutation in the Trp-tRNA synthetase affecting the Km for tryptophan, such that varying the exogenous concentration of tryptophan led to variation in the level of Trp-tRNA(Trp) in the cell. With this system, we found that the signal eliciting ppGpp synthesis during a stringent response triggered by tryptophan limitation did not depend on the absolute concentration of either charged or uncharged tRNA(Trp) but rather depended on a decline in the ratio of charged/uncharged tRNA(Trp). In addition, we found that the amplitude of the response, once triggered by tryptophan limitation, was determined by the total concentration of tRNA(Trp) present in the cell (which is mostly uncharged at that point in time). However, excess uncharged tRNA(Trp) did not amplify ppGpp synthesis triggered by limitation of a different amino acid. These data provide in vivo support for the in vitro-derived model of ppGpp synthesis on ribosomes.     

Conversion of a methionine initiator tRNA into a tryptophan-inserting elongator tRNA in vivo.

The role of the anticodon and discriminator base in aminoacylation of tRNAs with tryptophan has been explored using a recently developed in vivo assay based on initiation of protein synthesis by mischarged mutants of the Escherichia coli initiator tRNA. Substitution of the methionine anticodon CAU with the tryptophan anticodon CCA caused tRNA(fMet) to be aminoacylated with both methionine and tryptophan in vivo, as determined by analysis of the amino acids inserted by the mutant tRNA at the translational start site of a reporter protein containing a tryptophan initiation codon. Conversion of the discriminator base of tRNA(CCA)fMet from A73 to G73, the base present in tRNA(Trp), eliminated the in vivo methionine acceptor activity of the tRNA and resulted in complete charging with tryptophan. Single base changes in the anticodon of tRNA(CCA)fMet containing G73 from CCA to UCA, GCA, CAA, and CCG (changes underlined) essentially abolished tryptophan insertion, showing that all three anticodon bases specify the tryptophan identity of the tRNA. The important role of G73 in tryptophan identity was confirmed using mutants of an opal suppressor derivative of tRNA(Trp). Substitution of G73 with A73, C73, or U73 resulted in a large loss of the ability of the tRNA to suppress an opal stop codon in a reporter protein. Base pair substitutions at the first three positions of the acceptor stem of the suppressor tRNA caused 2-12-fold reductions in the efficiency of suppression without loss of specificity for aminoacylation of the tRNA with tryptophan.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of the trp RNA-binding attenuation protein (TRAP) with anti-TRAP

The trp RNA-binding attenuation protein (TRAP) negatively regulates expression of the tryptophan biosynthesis genes of Bacillus subtilis. In the presence of tryptophan, TRAP is activated to bind to the 5'-leader region of the trp mRNA resulting in termination prior to the structural genes. In addition, accumulation of uncharged tRNA(Trp) induces synthesis of anti-TRAP (AT), which binds to TRAP and inhibits its function. Both of these proteins consist of oligomers of identical subunits. Here, we characterize the self-association of each of these proteins and the TRAP-AT interaction in free solution using equilibrium and velocity analytical ultracentrifugation. TRAP exists as a stable 11-mer in the absence and in the presence of tryptophan. Tryptophan binding induces a conformational change in TRAP. AT exists in a reversible equilibrium between trimer and dodecamer with an equilibrium constant of approximately 3 x 10(14)M(-3). About 20% of the trimer is incompetent to form dodecamer. The AT equilibrium is slow on the time-scale of the velocity experiment. Formation of TRAP-AT complexes occurs only in the presence of tryptophan. A complex containing one TRAP 11-mer and one AT 12-mer forms with high affinity. At higher ratios of TRAP:AT complexes containing two TRAP 11-mers and one AT 12-mer are detected. A model for the structure of the complex is proposed.     

Resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants.

Single tryptophan mutants of the trp aporepressor, tryptophan 19-->phenylalanine (W19F) and tryptophan 99-->phenylalanine (W99F), were used in this study to resolve the individual steady-state and time-resolved fluorescence urea unfolding profiles of the two tryptophan residues in this highly intertwined, dimeric protein. The wild-type protein exhibits a large increase in fluorescence intensity and lifetime, as well as a large red shift in the steady-state fluorescence emission spectrum, upon unfolding by urea (Lane, A.N. & Jardetsky, O., 1987, Eur. J. Biochem. 164, 389-396; Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011-7020; Fernando, T. & Royer, C.A., 1992, Biochemistry 31, 6683-6691). Unfolding of the W19F mutant demonstrated that Trp 99 undergoes a large increase in intensity and a red shift upon exposure to solvent. Lifetime studies revealed that the contribution of the dominant 0.5-ns component of this tryptophan tends toward zero with increasing urea, whereas the longer lifetime components increase in importance. This lifting of the quenching of Trp 99 may be due to disruption of the interaction between the two subunits upon denaturation, which abolishes the interaction of Trp 99 on one subunit with the amide quenching group of Asn 32 on the other subunit (Royer, C.A., 1992, Biophys. J. 63, 741-750). On the other hand, Trp 19 is quenched in response to unfolding in the W99F mutant. Exposure to solvent of Trp 19, which is buried at the hydrophobic dimer interface in the native protein, results in a large red shift of the average steady-state emission.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between histidyl-tRNA level and protein synthesis rate in wild-type and mutant Chinese hamster ovary cells.

A preliminary investigation was carried out to determine how conditional lethal mutants affected in particular aminoacyl-tRNA synthetases may be used to study the role of tRNA charging levels in protein synthesis. The relationship between rate of protein synthesis and level of histidyl-tRNA in wild-type cultured Chinese hamster ovary cells was determined using the analogue histidinol to inhibit histidyl-tRNA synthetase activity. This response was compared with that obtained using a mutant strain with a defective histidyl-tRNA synthetase that phenotypically shows decreased rates of protein synthesis at reduced concentrations of histidine in the growth medium. The approach used was based on measuring the histidyl-tRNA levels in live cells. The percentage charging was estimated by comparing [14C]histidine incorporated into alkali-labile material in paired samples, one of which was treated with cycloheximide, five minutes before terminating during the incubation, to produce maximal aminoacylation. Wild-type cells under histidinol inhibition exhibited a sensitive, sigmoidal relationship between the level of histidyl-tRNA and the rate of protein synthesis. A decrease in the relative percentage of acylated tRNA (His) from 46% to 35% elicited a large reduction in the rate of protein synthesis from 90% to 30% relative to untreated cells. An unpredicted result was that the relationship between protein synthesis and histidyl-tRNA in the mutant was essentially linear. High acylation values for tRNA (His) were associated with rates of protein synthesis that were not nearly as high as in wild-type cells. These findings suggest that the charging charging levels of tRNA (His) isoacceptors could play a regulatory role in determining the rate of protein synthesis under conditions of histidine starvation in normal cells. The mutant appears to be a potentially useful system for studying the pivotal role of tRNA charging in protein synthesis, assuming that the altered response in the mutant is caused by its altered synthetase.     

Substrate specificity of a mutant alanyl-transfer ribonucleic acid synthetase of Escherichia coli

The correlation between the in vivo functioning and the in vitro behavior of the thermolabile alanyl-transfer ribonucleic acid (tRNA) synthetase (ARS) of Escherichia coli strain BM113 is presented. As a measure for the ARS activity inside the cell, the amount of acylated tRNA(ala) in vivo was determined. The rapid drop of the per cent tRNA(ala) charged which was observed upon shifting a culture of BM113 to the nonpermissive temperature indicates that in vivo acylation of tRNA(ala) might be the growth-limiting step at high temperature. Since neither growth nor the in vivo charging level of tRNA(ala) was affected by the addition of high l-alanine concentrations to the medium, one may infer that impaired functioning of the mutant enzyme at 40 C seems not to be due to reduced affinity of the enzyme for the amino acid. Separation of bulk tRNA of E. coli and of yeast on benzoylated diethylaminoethyl cellulose and charging of the fractions of the column by wild-type and mutant ARS reveal that only those tRNA species aminoacylated by the wild-type enzyme are also charged by the mutant ARS. Determination of the K(m) values of wild-type and mutant ARS for the three isoaccepting tRNA(ala) species of E. coli shows a ca. 10-fold increase of the apparent K(m) values of the mutant enzyme for all three species. Thus, the mutation proportionally reduces the apparent affinity for tRNA(ala) without causing any detectable recognition errors. Investigation of heat inactivation kinetics of wild-type and mutant ARS without and in the presence of substrates provides further evidence that only the transfer site of the ARS is altered by the mutation. Moreover, whereas both enzymes possess the same pH optimum of the relative maximal velocity, their pH dependence of the K(m) values for tRNA is different. The K(m) of the wild-type enzyme decreases at pH values below 7.0 and that of the mutant enzyme shows the inverse tendency; this again indicates an alteration of the tRNA binding site.     

Structural change in β-sheet A of Z α(1)-antitrypsin is responsible for accelerated polymerization and disease

The presence of the Z mutation (Glu342Lys) is responsible for more than 95% of α₁-antitrypsin (α₁AT) deficiency cases. It leads to increased polymerization of the serpin α₁AT during its synthesis and in circulation. It has been proposed that the Z mutation results in a conformational change within the folded state of antitrypsin that enhances its polymerization. In order to localize the conformational change, we have created two single tryptophan mutants of Z α₁AT and analyzed their fluorescence properties. α₁AT contains two tryptophan residues that are located in distinct regions of the molecule: Trp194 at the top of β-sheet A and Trp238 on β-sheet B. We have replaced each tryptophan residue individually with a phenylalanine in order to study the local environment of the remaining tryptophan residue in both M and Z α₁AT. A detailed fluorescence spectroscopic analysis of each mutant was carried out, and we detected differences in the emission spectrum, the Stern-Volmer constant for potassium iodide quenching and the anisotropy of only Trp194 in Z α₁AT compared to M α₁AT. Our data reveal that the Z mutation results in a conformational change at the top of β-sheet A but does not affect the structural integrity of β-sheet B.     

Zinc is required for assembly and function of the anti-trp RNA-binding attenuation protein,AT

The anti-TRAP protein (AT) of Bacillus subtilis regulates expression of the trp operon and other genes concerned with tryptophan metabolism. AT acts by inhibiting the tryptophan-activated trp RNA-binding attenuation protein (TRAP). AT is an oligomer of identical 53-residue polypeptides; it is produced in response to the accumulation of uncharged tRNA(Trp). Each AT polypeptide has two cysteine-rich clusters that correspond to the signature motif of the cysteine-rich zinc-binding domain of the chaperone protein DnaJ. Here we characterize the putative zinc-binding domain of AT and establish the importance of zinc for AT assembly and activity. AT is shown to contain Zn(II) at a ratio of one ion per monomer. Bound zinc is necessary for maintenance of the quaternary structure of AT; the removal of zinc converts the AT complex into inactive monomers. All four cysteine residues in the AT polypeptide are involved in Zn(II) coordination. Chemical cross-linking analyses indicate that the AT functional oligomer is a hexamer composed of two trimers. Substituting alanine for any cysteine residue of AT results in rapid degradation of the mutant protein in vivo. We propose a model for the AT trimer in which three AT chains are held together by three zinc atoms, each coordinated by the N-terminal segment and the C-terminal segment of separate AT polypeptides.     

Protein synthesis in hyperoxic endothelial cells: evidence for translational defect

To study the effects of hyperoxia on protein synthesis in primary cultures of porcine aortic endothelial cells, we exposed confluent cells to different O2 concentrations for various durations. Exposure to 95% O2 for 5 days resulted in a 71% inhibition of [3H]phenylalanine incorporation into total proteins. When compared with control cells, we observed no changes in 1) the pool size of free cytoplasmic phenylalanine and of phenylalanine attached to transfer RNA (tRNA), 2) the rate of protein degradation, and 3) the rate of charging of tRNA with phenylalanine. We found that under hyperoxic conditions 1) the incorporation of [3H]-uridine into total and polyadenylated RNA was increased, 2) the efficiency of extracted messenger RNA to direct protein synthesis in a reticulocyte lysate was maintained, 3) the proportion of polymeric to monomeric ribosomes was slightly increased, and 4) the rate of elongation, as measured by the ribosomal transit time, was decreased. Thus the reduction in protein synthesis in hyperoxic cells appears to result primarily from defects at the translational level in polypeptide chain elongation.     

Possible role of two phenylalanine residues in the active site of human cytidine deaminase

Site-directed mutagenesis on human cytidine deaminase (CDA) was employed to mutate specifically two highly conserved phenylalanine residues, F36 and F137, to tryptophan; at the same time, the unique tryptophan residue present in the sequence at position 113 was mutated to phenylalanine. These double mutations were performed in order to have for each protein a single tryptophan signal for fluorescence studies relative to position 36 or 137. The mutant enzymes thus obtained, W113F, F36W/W113F and F137W/W113F, showed by circular dicroism and thermal stability an overall structure not greatly affected by the mutations. The titration of Trp residues by N-bromosuccinimide (NBS) suggested that residue W113 of the wild-type CDA and W36 of mutant F36W/W113F are buried in the tertiary structure of the enzyme, whereas the residue W137 of mutant F137W/W113F is located near the surface of the molecule. Kinetic experiments and equilibrium experiments with FZEB showed that the residue W113 seems not to be part of the active site of the enzyme whereas the Phe/Trp substitution in F36W/W113F and F137W/W113F mutant enzymes had a negative effect on substrate binding and catalysis, suggesting that F137 and F36 of the wild-type CDA are involved in a stabilizing interaction between ligand and enzyme.