Regulation of E.coli phenylalanyl-tRNA synthetase operon in vivo

The phenylalanyl-tRNA synthetase operon is composed of two adjacent, cotranscribed genes, pheS and pheT, corresponding respectively to the small and large subunit of phenylalanyl-tRNA synthetase. A fusion between the regulatory regions of phenylalanyl-tRNA synthetase operon and the lac structural genes has been constructed to study the regulation of the operon. The pheS,T operon was shown, using the fusion, to be derepressed when phenylalanine concentrations were limiting in a leaky auxotroph mutated in the phenylalanine biosynthetic pathway. Furthermore, a mutational alteration in the phenylalanyl-tRNA synthetase gene, bradytrophic for phenylalanine, was also found to be derepressed under phenylalanine starvation. These results indicate that the pheS,T operon is derepressed when the level of tRNAPhe aminoacylation is lowered. By analogy with other well-studied amino acid biosynthetic operons known to be controlled by attenuation, these in vivo results indicate that phenylalanyl-tRNA synthetase levels are controlled by an attenuation-like mechanism.     

Regulation of E. coli phenylalanyl-tRNA synthetase operon in vivo

The phenylalanyl-tRNA synthetase operon is composed of two adjacent, cotranscribed genes, pheS and pheT, corresponding respectively to the small and large subunit of phenylalanyl-tRNA synthetase. A fusion between the regulatory regions of phenylalanyl-tRNA synthetase operon and the lac structural genes has been constructed to study the regulation of the operon. The pheS,T operon was shown, using the fusion, to be derepressed when phenylalanine concentrations were limiting in a leaky auxotroph mutated in the phenylalanine biosynthetic pathway. Furthermore, a mutational alteration in the phenylalanyl-tRNA synthetase gene, bradytrophic for phenylalanine, was also found to be derepressed under phenylalanine starvation. These results indicate that the pheS,T operon is derepressed when the level of tRNA^Phe aminoacylation is lowered. By analogy with other well-studied amino acid biosynthetic operons known to be controlled by attenuation, these in vivo results indicate that phenylalanyl-tRNA synthetase levels are controlled by an attenuation-like mechanism.     

Defective regulation of the phenylalanine biosynthetic operon in mutants of the phenylalanyl-tRNA synthetase operon.

Among mutants of Escherichia coli resistant to p-fluorophenylalanine (PFP) were some with constitutive expression of the phenylalanine biosynthetic operon (the pheA operon). This operon is repressed in the wild type by phenylalanine. The mutation in three of these mutants mapped in the aroH-aroD region of the E. coli chromosome at 37 min. A plasmid bearing wild-type DNA from this region restored p-fluorophenylalanine sensitivity and wild-type repression of the pheA operon. Analysis of subclones of this plasmid and comparison of its restriction map with published maps indicated that the mutations affecting regulation of the pheA operon lie in the structural genes for phenylalanyl-tRNA synthetase, pheST, probably in pheS. Thus, the pheST operon has a role in the regulation of phenylalanine biosynthesis, the most likely being that wild-type phenylalanyl-tRNA synthetase maintains a sufficient intracellular concentration of Phe-tRNA(Phe) for attenuation of the pheA operon in the presence of phenylalanine. A revised gene order for the 37-min region of the chromosome is reported. Read clockwise, the order is aroD, aroH, pheT, and pheS.     

Open reading frames in the control regions of the phenylalanyl-tRNA synthetase operon of E. coli

The pheST operon codes for the two subunits of phenylalanyl-tRNA synthetase and it expression is controlled by attenuation in a way similar to many amino acid biosynthetic operons. The nucleotide sequence of the control regions of the operon indicates the presence of several open reading frames besides that of the leader peptide. One of these open reading frames, called the alternative leader peptide, starts at about the same place as the leader peptide and ends after the terminator of the attenuator. Another open reading frame, called the terminator peptide, starts after the terminator and covers about half the distance to pheS, the first structural gene of the operon. The present report shows that, in fact, the only open reading frame to be translated efficiently is the leader peptide itself. The alternative leader peptide and the terminator peptide are both translated at a negligible rate.     

Site-directed mutagenesis of the phosphate-binding consensus sequence in Escherichia coli adenylosuccinate synthetase.

Adenylosuccinate synthetases from different sources contain an N-terminal glycine-rich sequence GDEGKGK, which is homologous to the conserved sequence GXXXXGK found in many other guanine nucleotide-binding proteins or enzymes. To determine the role of this sequence in the structure and function of Escherichia coli adenylosuccinate synthetase, site-directed mutagenesis was performed to generate five mutant enzymes: G12V (Gly12----Val), G15V (Gly15----Val), G17V (Gly17----Val), K18R (Lys18----Arg), and I19T (Ile19----Thr). Comparison of the kinetic properties of the wild-type enzyme and those of the mutant enzymes revealed that the sequence is critical for enzyme activity. Replacement of Gly12, Gly15, or Gly17 with Val, or replacement of Lys18 with Arg, resulted in significant decreases in the kcat/Km values of the enzyme. Because the consensus sequence GXXXXGK(T/S) has been found in many GTP-binding proteins, isoleucine at position 19 in the E. coli adenylosuccinate synthetase was changed to threonine to produce the sequence GDEGKGKT. This mutation, which more closely resembles the consensus sequence, resulted in a 160-fold increase in the Km value for substrate GTP; however, there were no great changes for the other two substrates, IMP and aspartate. Based on these data, we suggest that the N-terminal glycinerich sequence in E. coli adenylosuccinate synthetase plays a more important role in enzyme catalysis than in substrate binding. In addition, a hydrophobic amino acid residue such as isoleucine, leucine, or valine, rather than threonine, may play a critical role in GTP binding in adenosuccinate synthetase. These findings suggest that the glycine-rich sequence in adenylosuccinate synthetase functions differently relative to those in other GTP binding proteins or enzymes.     

The regulatory region of the L-arabinose operon: a physical, genetic and physiological study.

A fine-structure physical and genetic map was constructed of a 1000 base-pair region of the l-arabinose operon of Escherichia coli. This region consists of the ara regulatory sequences contained between the araC and araB genes and portions of these flanking genes. Point mutations, Mu phage insertions, and bacterial deletions as well as arabinose-induced and basal enzyme levels in the strains were used in constructing a genetic map of the region. These ordered positions were then located more accurately by mapping the point mutations against physically located endpoints of deletions isolated on the two non-defective transducing phage λparaB114 and λparaC116. Phage possessing deletions ending in the arabinose regulatory region were isolated from indicating-plates on which deletions removing none, part, or all of either the araC or araB genes carried on the phage could be distinguished. Phage stocks were enriched for such deletions prior to plating by treatment with chelating agents and heat (Parkinson &; Huskey, 1971). Deletions into the ara region on either phage shorten the ara DNA homology region formed from heteroduplexes between λparaB114 and λparaB116. Therefore the physical locations of these deletion endpoints were determined by electron microscopy of the appropriate heteroduplexes and/or by gel electrophoresis of the central duplex following S₁ nuclease digestion (Lis &; Schleif, 1975b). 18 of the 32 deletions isolated and mapped in this region were measured physically. The space between araC and araB, containing the regulatory elements of the operon, is estimated to be about 300 base-pairs.     

tRNA discrimination by T. thermophilus phenylalanyl-tRNA synthetase at the binding step

The extent of tRNA recognition at the level of binding by Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS), one of the most complex class II synthetases, has been studied by independent measurements of the enzyme association with wild-type and mutant tRNA(Phe)s as well as with non-cognate tRNAs. The data obtained, combined with kinetic data on aminoacylation, clearly show that PheRS exhibits more tRNA selectivity at the level of binding than at the level of catalysis. The anticodon nucleotides involved in base-specific interactions with the enzyme prevail both in the initial binding recognition and in favouring aminoacylation catalysis. Tertiary nucleotides of base pair G19-C56 and base triple U45-G10-C25 contribute primarily to stabilization of the correctly folded tRNA(Phe) structure, which is important for binding. Other nucleotides of the central core (U20, U16 and of the A26-G44 tertiary base pair) are involved in conformational adjustment of the tRNA upon its interaction with the enzyme. The specificity of nucleotide A73, mutation of which slightly reduces the catalytic rate of aminoacylation, is not displayed at the binding step. A few backbone-mediated contacts of PheRS with the acceptor and anticodon stems revealed in the crystal structure do not contribute to tRNA(Phe) discrimination, their role being limited to stabilization of the complex. The highest affinity of T. thermophilus PheRS for cognate tRNA, observed for synthetase-tRNA complexes, results in 100-3000-fold binding discrimination against non-cognate tRNAs.     

Site-directed mutagenesis of proline 204 in the 'hinge' region of yeast phosphoglycerate kinase.

Site-specific mutants have been produced in order to investigate the role of proline 204 in the 'hinge' region of yeast phosphoglycerate kinase (PGK). This totally conserved proline has been shown to be the only cis-proline in the high resolution crystal structures of yeast, B. stearothermophilus, T. brucei and T. maritima PGK, and may therefore have a role in the independent folding of the two domains or in the 'hinge' bending of the molecule during catalysis. The residue was replaced by a histidine (Pro204His) and a phenylalanine (Pro204Phe), and the resulting proteins characterised by differential scanning calorimetry (DSC), circular dichroism (CD), tryptophan fluorescence emission and kinetic analysis. Although the secondary and tertiary structure of the Pro204His protein is generally similar to that of the wild-type enzyme as assessed by CD, the enzyme is less stable to heat and guanidinium chloride denaturation than the wild-type. In the denaturation experiments two transitions were observed for both the wild-type and the Pro204His mutant, as have been previously reported for yeast PGK [Missiakas, D., Betton, J.M., Minard, P. & Yon, J.M. (1990) Biochemistry 29, 8683-8689]. The first transition is accompanied by an increase in fluorescence intensity leading to a hyperfluorescent state, followed by the second, corresponding to a decrease in fluorescence intensity. However, for the Pro204His mutant, the first transition proceeded at lower concentrations of guanidinium chloride and the second transition proceeded to the same extent as for the wild-type protein, suggesting that sequence-distant interactions are more rapidly disrupted in this mutant enzyme than in the wild-type enzyme, while sequence-local interactions are disrupted in a similar way. The Michaelis constants (K(m)) for both 3-phospho-D-glycerate and ATP are increased only by three or fourfold, which confirms that, as expected, the substrate binding sites are largely unaffected by the mutation. However, the turnover and efficiency of the Pro204His mutant is severely impaired, indicating that the mechanism of 'hinge' bending is hindered. The Pro204Phe enzyme was shown to be significantly less well folded than the wild-type and Pro204His enzymes, with considerable loss of both secondary and tertiary structure. It is proposed that the proline residue at 204 in the 'hinge' region of PGK plays a role in the stability and catalytic mechanism of the enzyme.     

Interaction of T. thermophilus phenylalanyl-tRNA synthetase with the 3'-terminal nucleotide of tRNAPhe

The interaction of Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) with the 3;-terminal nucleotide of tRNAPhe has been studied by affinity labeling to solve the problem arising from X-ray crystallographic study: the binding sites of phenylalanine and the 3;-terminal nucleotide base were revealed to be identical in the crystal structures of PheRS complexed with the substrates. tRNAPhe derivatives containing a photoreactive 4-thiouridine (tRNAPhe-s4U-76) or 6-thioguanosine residue (tRNAPhe-s6G-76) in the 3;-end have been prepared using terminal tRNA nucleotidyl transferase. Kinetic measurements of aminoacylation provide evidence for a functional role of base-specific interactions of the 3;-terminal adenosine in productive interaction of tRNAPhe with the enzyme: tRNAPhe-s4U-76 cannot be aminoacylated; the replacement of A-76 with s6G results in a 370-fold reduction of catalytic efficiency of aminoacylation mainly due to decreased Vmax value. Relative cross-linking of the s6G-substituted tRNA to the alpha-subunit (69% of the total yield of the cross-linked alpha- and beta-subunits) is two times higher as compared to the cross-linking of tRNAPhe-s4U-76. The dialdehyde derivative, tRNAPhe-Aox-76, with periodate-oxidized 3;-terminal ribose is cross-linked with the same selectivity to the alpha-subunit as tRNAPhe-s6G-76. The results suggest specific binding of the 3;-terminal nucleotide of tRNAPhe by the catalytic subunit of PheRS in the absence of other substrates. Comparative analysis of the cross-linked products in the absence and in the presence of small substrates revealed ATP and aminoacyl-adenylate to effect the interaction of the tRNAPhe acceptor end with PheRS. The correct positioning of the 3;-terminal nucleotide of tRNAPhe corresponding to the structure of the productive complex with PheRS is therefore promoted only in the presence of all three substrates.     

Site-directed mutagenesis of alpha-tubulin. Reductive methylation studies of the Lys 394 region.

Previous studies have implicated at least two regions in alpha-tubulin that are important for the regulation of microtubule assembly. These regions include a cluster of basic residues consisting of Arg 390, His 393, and Lys 394 and the highly acidic carboxyl terminus. Lys 394 is highly reactive to HCHO and NaCNBH3. The reductive methylation of Lys 394 by these reagents is thought to be responsible for the profound inhibitory effects of low concentrations of HCHO on microtubule assembly (cf. Szasz J., M. B. Yaffe, M. Elzinga, G. S. Blank, and H. Sternlicht. 1986. Biochemistry. 25:4572-4582). In this study we reexamined the basis for this inhibition. Lys 394 in a human keratinocyte alpha-tubulin (k alpha 1) was replaced by a glutamic acid residue using site-directed mutagenesis. The mutant K394E was synthesized in vitro using rabbit reticulocyte lysates, and its ability to coassemble with bovine brain microtubule protein (MTP) before and after reaction with HCHO and NaCNBH3 was compared with that of wild-type. No differences in the coassemblies of the unmethylated proteins were detected suggesting that Lys 394 is not essential for microtubule assembly. However, methylated K394E prepared at low HCHO concentrations (< 1 mM) incorporated into microtubules to a greater extent (approximately 30-40%) than methylated wild-type. This result is consistent with the hypothesis that methylation of Lys 394 interferes with microtubule assembly. However, the extent of protection afforded by the replacement of Lys 394 with Glu 394 was less than half as large as that predicted from the earlier studies. We tentatively conclude that another residue(s) besides Lys 394 contributes significantly to the assembly-inhibition observed with low concentrations of HCHO. Since this residue(s) is less reactive than Lys 394, it would have to inhibit assembly substoichiometrically when methylated. Potential candidates for this residue include bulk lysyl residue(s), a lysyl residue(s) with intermediate reactivity toward HCHO, and the NH2-termini. The NH2-termini are especially attractive candidates since they appear to have a structural role in microtubule assembly.     

Site-directed mutagenesis of the regulatory domain of Escherichia coli carbamoyl phosphate synthetase identifies crucial residues for allosteric regulation and for transduction of the regulatory signals

Carbamoyl phosphate (CP), the essential precursor of pyrimidines and arginine, is made in Escherichia coli by a single carbamoyl phosphate synthetase (CPS) consisting of 41.4 and 117.7 kDa subunits, which is feed-back inhibited by UMP and activated by IMP and ornithine. The large subunit catalyzes CP synthesis from ammonia in three steps, and binds the effectors in its 15 kDa C-terminal domain. Fifteen site-directed mutations were introduced in 13 residues of this domain to investigate the mechanism of allosteric modulation by UMP and IMP. Two mutations, K993A and V994A, decreased significantly or abolished enzyme activity, apparently by interfering with the step of carbamate synthesis, and one mutation, T974A, negatively affected ornithine activation. S948A, K954A, T974A, K993A and K993W/H995A abolished or greatly hampered IMP activation and UMP inhibition as well as the binding of both effectors, monitored using photoaffinity labeling and ultracentrifugation binding assays. V994A also decreased significantly IMP and UMP binding. L990A, V991A, H995A, G997A and G1008A had more modest effects or affected more the modulation by and the binding of one than of the other nucleotide. K993W, R1020A, R1021A and K1061A were without substantial effects. The results confirm the independence of the regulatory and catalytic centers, and also confirm functional predictions based on the X-ray structure of an IMP-CPS complex. They prove that the inhibitor UMP and the activator IMP bind in the same site, and exclude that the previously observed binding of ornithine and glutamine in this site were relevant for enzyme activation. K993 and V994 appear to be involved in the transmission of the regulatory signals triggered by UMP and IMP binding. These effectors possibly change the position of K993 and V994, and alter the intermolecular contacts mediated by the regulatory domain.     

Site-directed mutagenesis and expression of PC2 in microinjected Xenopus oocytes.

The biosynthesis and post-translational maturation of PC2, a neuroendocrine-specific Kex2-like endoprotease, following expression in Xenopus oocytes is described. The initial translation product was a 75-kDa membrane-associated protein which was released from the oocytes as a glycosylated 71-kDa protein. During extended chase periods, the extracellular 71-kDa protein was converted to a mature 68-kDa product. A deletion mutant lacking a putative COOH-terminal amphipathic helix was still membrane-associated, suggesting that this domain was not essential for attachment of PC2 to membranes. Two putative proregion cleavage site mutants were also constructed. Conversion of the 75-kDa peptide to the 71-kDa peptide involved cleavage at the sequence Lys-Arg-Arg-Arg (amino acids 78-81), since mutation of this sequence to Lys-Val-Arg-Leu resulted in the secretion of the 75-kDa peptide. Extracellular conversion of the 71-kDa peptide to the 68-kDa peptide involved cleavage at the sequence Arg-Lys-Lys-Arg (amino acids 106-109), since deletion of this tetrabasic sequence resulted in secretion of the 71-kDa peptide without further conversion to the 68-kDa form. Finally, a mutation which changed a catalytically important Asp to Asn did not affect processing of proPC2. These results may be relevant to our understanding of mechanisms in the intracellular sorting and maturation of proPC2 in neuroendocrine cells.     

Yeast phenylalanyl-tRNA synthetase. Properties of the histidyl residues.

Reactivity of the histidyl groups of yeast phenylalanyl-tRNA synthetase was studied in the absence or presence of substrates. In the absence of substrates about 10 histidine residues were found to react with similar kinetic constants. Phenylalanine at 10(-3) M was found to protect two histidyl residues; increasing the amino acid concentration to 5 . 10(-3) M resulted in the protection of two more histidyl groups. tRNAPhe did not afford any protection to histidine residues, but acylated phenylalanyl-tRNA (Phe-tRNAPhe) protected two of the four histidyl groups already protected by phenylalanine. These results suggest the existence of two different sets of accepting sites for phenylalanine: one specific for the free amino acid, the other one specific for the amino acid linked to the tRNA, but being accessible to free phenylalanine, with a somewhat lower binding constant, ATP was found to mask around four histidyl residues against diethylpyrocarbonate modification. By photoirradiation of enzyme-phenylalanine complex in the presence of rose bengale, a significant amount of amino acid was bound to the alpha subunit (Mr = 73 000) of phenylalanyl-tRNA synthetase, confirming that the amino acid binding site is located on this subunit, as previously suggested by modification of thiol groups. Upon irradiation of an enzyme-tRNA complex, almost no covalent binding of tRNA occurred during enzyme inactivation, suggesting that the histidyl residues involved in the enzymic activity are not required for tRNA binding.