Reversion of trpA nonsense mutations by deletion of the chain-termination codons

This paper describes a novel mechanism for reversion of nonsense mutations in the trpA gene of Escherichia coli. This mechanism, deletion of the nonsense codon, was discovered in the course of selecting for missense revertants of trpA(UGA211) and for catalytically active tryptophan synthetase alpha chain revertants of trpA(UAA234) and trpA(UAG234). Each type of revertant trpA was cloned and its DNA sequence determined. trpA(UGA211) gave rise to two previously unidentified types of missense revertant. The first type was expected, namely trpA(CGA211), the result of a base substitution event. The other type, representing approximately 1% of the missense revertants, was unexpected on the basis of single base substitutions and an understanding of which amino acids are functional at alpha chain position 211. It was found to be the result of a 21 base-pair deletion of a region containing codon 211. The tryptophan-independent revertants of both position 234 nonsense mutants occurred at a frequency of approximately 2 per 10(9) viable cells. They were identical in that they both resulted from a 3 base-pair deletion, namely deletion of the chain-terminating codon at position 234. One of them, however, also displayed an A instead of the normal G in the third position of codon 235. The revertants were characterized according to growth in different media and tryptophan synthetase assays performed on crude extracts. These types of mutants should prove interesting and important for the elucidation of alpha chain structure-function relationships, for insight into the assembly and interaction of subunits in this model multienzyme complex, and for the study of mechanisms by which deletions can be generated.     

Relative activities and stabilities of mutant Escherichia coli tryptophan synthase alpha subunits

In vitro mutagenesis of the Escherichia coli trpA gene has yielded 66 mutant tryptophan synthase alpha subunits containing single amino acid substitutions at 49 different residue sites and 29 double and triple amino acid substitutions at 16 additional sites, all within the first 121 residues of the protein. The 66 singly altered mutant alpha subunits encoded from overexpression vectors have been examined for their ability to support growth in trpA mutant host strains and for their enzymatic and stability properties in crude extracts. With the exception of mutant alpha subunits altered at catalytic residue sites Glu-49 and Asp-60, all support growth; this includes those (48 of 66) that have no enzymatic defects and those (18 of 66) that do. The majority of the enzymatically defective mutant alpha subunits have decreased capacities for substrate (indole-3-glycerol phosphate) utilization, typical of the early trpA missense mutants isolated by in vivo selection methods. These defects vary in severity from complete loss of activity for mutant alpha subunits altered at residue positions 49 and 60 to those, altered elsewhere, that are partially (up to 40 to 50%) defective. The complete inactivation of the proteins altered at the two catalytic residue sites suggest that, as found via in vitro site-specific mutagenesis of the Salmonella typhimurium tryptophan synthetase alpha subunit, both residues probably also participate in a push-pull general acid-base catalysis of indole-3-glycerol phosphate breakdown for the E. coli enzyme as well. Other classes of mutant alpha subunits include some novel types that are defective in their functional interaction with the other tryptophan synthetase component, the beta 2 subunit. Also among the mutant alpha subunits, 19 were found altered at one or another of the 34 conserved residue sites in this portion of the alpha polypeptide sequence; surprisingly, 10 of these have wild-type enzymatic activity, and 16 of these can satisfy growth requirements of a trpA mutant host. Heat stability and potential folding-rate alterations are found in both enzymatically active and defective mutant alpha subunits. Tyr-4. Pro-28, Ser-33, Gly-44, Asp-46, Arg-89, Pro-96, and Cys-118 may be important for these properties, especially for folding. Two regions, one near Thr-24 and another near Met-101, have been also tentatively identified as important for increasing stability.     

Suppression of Glutamic Acid Codons by Mutant Glycine Transfer Ribonucleic Acid

In previous mutational studies with mutant trpA46 (Gly [GGA] --> Glu [GAA] at position 211 of the tryptophan synthetase alpha chain) of Escherichia coli, no missense suppressors were detected. Such suppressors have now been obtained by single mutations in gly Vins, the structural gene for a GGA/G-reading, mutationally altered form of gly V transfer ribonucleic acid (tRNA) (tRNA(Gly) which reads GGU/C). A trpA46 strain containing the gly Vins alteration was mutagenized with hydroxylamine, and suppressor mutations were detected in the prototrophs obtained. Eighteen independent suppressors were examined and shown to have alterations which map in the gly V region. Chromatography of the glycyl-tRNAs of one suppressed mutant on a benzoylated diethylaminoethyl-cellulose column revealed an alteration in the tRNA(ins) (Gly) peak. The trpA46 suppressor mutation thus appears to involve a change of tRNA(ins) (Gly) from a GGA/G (Gly) reader to a GAA (Glu) reader. Since this suppressor presumably retains the "wobble" pairing of gly Vins tRNA, it was used to select the conversion of GAU (Asp211) to GAG (Glu211) in the alpha chain. supD (serine-inserting amber suppressor) was then used to obtain the conversion of GAG (Glu211) to UAG211. Missense revertants of trpA (UAG211) are being isolated as a means of introducing new codons which can be used in the selection of additional missense suppressors.     

Tryptophan synthetase alpha(5.7-S): novel molecular species formed within Escherichia coli.

A novel molecular species contributes about 5% of the total tryptophan synthetase of Escherichia coli derepressed for the trp operon enzymes. The new species is identified under conditions in which the dissociation of the two nonidentical subunits of the tryptophan synthetase complex is favored. The new species sediments at 5.7S, catalyzes the conversion of indole-3-glycerol phosphate to indole, and has been designated alpha(5.7-S). Although alpha(5.7-S) is not observed in extracts of trpA or trpB mutant strains deficient in the ability to form tryptophan synthetase alpha or beta2 subunits, respectively, a mixture of the two extracts allows the formation of alpha(5.7-S). Similar results are obtained when a homogeneous alpha protein is mixed with an extract of a trpA mutant strain, suggesting that the interaction of alpha and beta2 proteins is obligatory for alpha(5.7-S) formation. One can obtain a beta2 protein preparation that when mixed with a pure alpha protein gives no alpha(5.7-S). Therefore, the interaction of alpha and beta2 proteins alone is not sufficient for the formation of alpha(5.7-S). When a mixture of alpha and beta2 proteins devoid of alpha(5.7-S) is added to extracts of trp deletion mutants, the novel species can be reconstituted in vitro only when deletions are used that carry at least the operator-proximal part of the trpB gene. Therefore, it is concluded that the alpha(5.7-S) species of tryptophan synthetase results from the interaction of the alpha protein, the beta2 protein, and a third component, beta', specified by the deoxyribonucleic acid defined by the end points of two trp deletion mutants.     

Structural Interactions Between Amino Acid Residues at Positions 22 and 211 in the Tryptophan Synthetase Alpha Chain of Escherichia coli

Construction and characterization of double mutants altered in the structural gene of the tryptophan synthetase alpha chain of Escherichia coli revealed interactions between amino acid residues at positions 22 and 211. These interactions are specific for the particular amino acid residue at position 211. The results indicate also that amino acid residues which appear to be functionally near-equivalent in one configuration may strongly influence the activity of a protein with a subsequent change at another site. Seven independent suppressors of trpA218 (Leu22-Ser211) were isolated. Their properties suggest that all seven may suppress the codon (AGU/C) for Ser211. Six of the seven are co-transducible with glyV, the structural gene for the GGU/C-specific tRNA(Gly).     

Mitomycin C-induced expression of trpA of Salmonella typhimurium inserted into the plasmid ColE1.

EcoRI endonuclease digestion of the deoxyribonucleic acid of a phi80 transducing phage carrying the entire tryptophan (trp) operon of Salmonella typhimurium (phi80 S.t.trpE-A) yielded a 4.3 X 10(6)-dalton fragment containing intact trpE, trpD, and trpC and a 3.35 X 10(6)-dalton fragment containing intact trpA. The trpA fragment inserted into EcoRI-cleaved plasmids ColE1 and CR1 was expressed regardless of its orientation of insertion. Mitomycin C, a compound that induces colicin E1 production in ColE1-containing bacteria, stimulated tryptophan synthetase alpha production in cells containing ColE1-TRPA plasmids with the trpA fragment inserted in one orientation but not the other. We conclude that in the inducible plasmids trpA can be expressed from the colicin E1 promoter.     

Protein synthesis in Escherichia coli with mischarged tRNA

Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNA(Asp), while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNA(Asn), a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). On the basis of the E. coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNA(Asn) and its acceptance by elongation factor EF-Tu. While large amounts of Asp-tRNA(Asn) are detrimental to E. coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase.     

Characterization of missense suppressors of a double mutant of the tryptophan synthetase alpha chain of Escherichia coli.

Summary Eight suppressors of trpA218, a missense double mutant of trpA, the gene for the tryptophan synthetase alpha chain of Escherichia coli, have been further characterized genetically, physiologically and biochemically. trpA218 possesses an inactive alpha chain that contains leucine (instead of phenylalanine) at position 22 and serine (instead of glycine) at position 211. Replacement of either mutant amino acid by the corresponding wild type amino acid leads to an active alpha chain. To determine whether each trpA218 suppressor (Su218) affects the 22 or 211 position, a substitute trpA218 was constructed. Whereas the original double mutant possesses a Ser211 specified by the codon AGU, we constructed a trpA(Leu22-Ser211) in which the Ser211 codon is UCG. All eight Su218s failed to suppress the new double mutant. The suppressors fall into two classes according to growth in various media. Six of the eight map in the region of glyV, a gene for the GGU/C-reading glycine tRNA. After reversed phase column chromatography of radioactively labeled glycyl-tRNA, the suppressor tRNAs exhibited altered profiles that were similarly different from the parental tRNA in all eight cases. These results suggest that there are several classes of Su218, that all of them suppress the serine codon AGU (or AGC) corresponding to position 211, and that at least six of the eight are mutationally altered glycine tRNAs.A preliminary report of portions of this work was presented at the spring meeting of the Texas Branch of the American Society for Microbiology, College Station, Texas, March, 1975     

Tryptophan synthetase: its charmed history

Tryptophan synthetase was initially selected as a subject for investigation of the relationship between gene structure and protein structure. Early studies with this enzyme first demonstrated the existence in mutants of immunologically cross-reacting material (CRM) and the restoration of a wild-type enzyme by genetic suppression. Fine structure analyses with E. coli tryptophan synthetase missense mutants proved the colinearity of gene structure and catalytic capabilities of this enzyme have been subjects for numerous studies.     

The regulation of tryptophan biosynthesis in Pseudomonas aeruginosa

Summary Eighteen auxotrophs of Pseudomonas aeruginosa requiring l-tryptophan for growth were isolated following nitrosoguanidine mutagenesis. Mutant blocks for each step of tryptophan biosynthesis were identified by enzymological assay. A regulatory mutant was characterized which was simultaneously constitutive for the gene products of trpA, trpB and trpD. Another class of regulatory mutant appears to synthesize tryptophan synthetase (i.e., trpE and trpF subunits) constitutively. The results implicate three control entities in the pathway of tryptophan biosynthesis: (i) The gene products of trpA, trpB and trpD are repressible by tryptophan, the range of enzyme specific activity varying at least fifty-fold. (ii) No regulation of the trpC gene product could be demonstrated, indicating that its synthesis is constitutive. (iii) The gene products of rpE and trpF are inducible by indoleglycerol 3-phosphate; the magnitude of induction can exceed 100-fold. These results together with some genetic data indicate a general similarity in gene-enzyme relationships between P. aeruginosa and P. putida. A number of specific differences that distinguish the two species are noted.A mutant blocked in the common pathway of aromatic biosynthesis was used to prove that enzymes of tryptophan biosynthesis other than tryptophan synthetase are not inducible by precursors of the common pathway such as chorismate. It is concluded that the concentration of tryptophan that signals total repression of the gene products of trpA, trpB and trpD is lower than the concentrations necessary for maximal feedback inhibition of anthranilate synthetase and for abolition of the induction of tryptophan synthetase.     

Mapping of the tryptophan genes of Acinetobacter calcoaceticus by transformation

Auxotrophs of Acinetobacter calcoaceticus blocked in each reaction of the synthetic pathway from chorismic acid to tryptophan were obtained after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. One novel class was found to be blocked in both anthranilate and p-aminobenzoate synthesis; these mutants (trpG) require p-aminobenzoate or folate as well as tryptophan (or anthranilate) for growth. The loci of six other auxotrophic classes requiring only tryptophan were defined by growth, accumulation, and enzymatic analysis where appropriate. The trp mutations map in three chromosomal locations. One group contains trpC and trpD (indoleglycerol phosphate synthetase and phosphoribosyl transferase) in addition to trpG mutations; this group is closely linked to a locus conferring a glutamate requirement. Another cluster contains trpA and trpB, coding for the two tryptophan synthetase (EC 4.2.1.20) subunits, along with trpF (phosphoribosylanthranilate isomerase); this group is weakly linked to a his marker. The trpE gene, coding for the large subunit of anthranilate synthetase, is unlinked to any of the above. This chromosomal distribution of the trp genes has not been observed in other organisms.     

Temperature-sensitive repression of the tryptophan operon in Escherichia coli

Mutants of Escherichia coli exhibiting temperature-sensitive repression of the tryptophan operon have been isolated among the revertants of a tryptophan auxotroph, trpS5, that produces an altered tryptophanyl transfer ribonucleic acid (tRNA) synthetase. Unlike the parental strain, these mutants grew in the absence of tryptophan at high but not at low temperature. When grown at 43.5 C with excess tryptophan (repression conditions), they produced 10 times more anthranilate synthetase than when grown at 36 C or lower temperatures. Similar, though less striking, temperature-sensitivity was observed with respect to the formation of tryptophan synthetase. Transduction mapping by phage P1 revealed that these mutants carry a mutation cotransducible with thr at 60 to 80%, in addition to trpS5, and that the former mutation is primarily responsible for the temperature-sensitive repression. These results suggest that the present mutants represent a novel type of mutation of the classical regulatory gene trpR, which probably determines the structure of a protein involved in repression of the tryptophan operon. In agreement with this conclusion, tRNA of several trpR mutants was found to be normal with respect to its tryptophan acceptability. It was also shown that the trpS5 allele, whether present in trpR or trpR(+) strains, produced appreciably higher amounts of anthranilate synthetase than the corresponding trpS(+) strains under repression conditions. This was particularly true at higher temperatures. These results provide further evidence for our previous conclusion that tryptophanyl-tRNA synthetase is somehow involved in repression of this operon.     

A ribosome binding site sequence is necessary for efficient expression of the distal gene of a translationally-coupled gene pair

Expression of trpB and trpA of the Escherichia coli tryptophan operon is shown to be "translationally coupled", i.e., efficient translation of the trpA coding region is dependent on prior translation of the trpB coding region and termination of translation at the trpB stop codon. To examine the dependence of trpA expression on the ribosome binding site sequence in the distal segment of trpB, deletions were produced that replaced this trpB sequence. Analysis of trpA expression in these deletion mutants established that the ribosome binding site sequence is required for efficient translation of the trpA segment of trp mRNA. A modest effect of translation over the trpA ribosome binding site on independent initiation at that site was also observed.     

Fusion of trpB and trpA of Escherichia coli yields a partially active tryptophan synthetase polypeptide

The separate alpha and beta polypeptides of the tryptophan synthetase of bacteria are represented in fungi by a fusion polypeptide in which the first third is homologous to bacterial alpha chains and the remainder is homologous to bacterial beta chains. In the yeast polypeptide, a short nonhomologous "connector" joins the two homologous segments. The chromosomal order of all bacterial genes that specify tryptophan synthetase beta and alpha chains, respectively, is trpB-trpA. Fusion of these genes in their present arrangement would result in the synthesis of a polypeptide with a segmental order, N-beta-alpha-C, opposite that observed in fungi. To investigate possible explanations for the apparent transposition that occurred in the evolution of the fungal gene we have made two fusions of trpB and trpA of Escherichia coli in their natural orientation. We find that the fusion proteins are synthesized but both are less active catalytically than the wild type bacterial protein. In addition, the fusion proteins associate abnormally, they are activated only slightly by wild type alpha or beta 2, and they are less sensitive than the wild type protein to inhibition by antibodies to alpha or beta 2. The fusion proteins have normal substrate affinities. Our findings suggest that the altered structures of the fusion proteins affect catalytic ability and the locations of the alpha and/or beta chain combining sites. This structural distortion may have prevented the natural selection of direct gene fusions during the course of the fungal gene's evolution.     

Nucleotide sequences and genomic constitution of five tryptophan genes of Lactobacillus casei

Five trp genes, trpD, trpC, trpF, trpB, and trpA, of Lactobacillus casei were cloned by transformation of tryptophan auxotrophic mutants of the respective trp genes in Escherichia coli. These trp genes appear to constitute an operon and are located in the above order in a segment of DNA of 6,468 base pairs. The entire nucleotide sequence of this DNA segment was determined. Five contiguous open reading frames in this segment can encode proteins consisting of 341, 260, 199, 406, and 266 amino acids, respectively, in the same direction. The amino acid sequences of these proteins exhibit 25.5-50.2% homology with the amino acid sequences of the corresponding trp enzymes of E. coli. Two trp genes, trpC and trpF, from L. casei can complement mutant alleles of the corresponding genes of E. coli. However, neither the trpA gene nor the trpB gene of L. casei can complement mutations in the E. coli trpA gene and the trpB gene, respectively, suggesting that the protein products of the L. casei and E. coli trpA and trpB genes, respectively, cannot form heterodimers of tryptophan synthetase with activity. Other features of the coding and flanking regions of the trp genes are also described.     

Tryptophan biosynthesis in Salmonella typhimurium: location in trpB of a genetic difference between strains LT2 and LT7.

Salmonella typhimurium prototrophs carrying a trpR mutation synthesize tryptophan biosynthetic enzymes constitutively. When feedback inhibition of anthranilate synthetase but not 5'-phosphoribosylpyrophosphate phosphoribosyltransferase activity was by-passed by growing cells on media supplemented with anthranilic acid, all trpR prototrophs overproduced and excreted tryptophan. However, the rate of tryptophan production depended on both the ancestry of the trpR strain and the integrity of its trpA gene. Prototrophs with trp genes derived from S. typhimurium strain LT2 produced tryptophan more efficiently than those with trp genes derived from strain LT7. This strain difference was cryptic insofar as it did not affect the growth rate; it was revealed only as a rate-limiting step in the constitutive biosynthesis of tryptophan in the presence of anthranilic acid, and was due to a lesion in the LT7-derived trpB gene. Strains with LT7-derived trp genes bearing a deletion in trpA produced tryptophan as readily as LT2 trpR prototrophs. This indicated that LT7-specific 5-phosphoribosylpyrophosphate phosphoribosyltransferase must be aggregated with the trpA gene produce to give an observable reduction of constitutive tryptophan production. The discovery of this strain difference has particular implications for studies involving the activities of trpA and B genes and their products in S. typhimurium and may have general significance for other studies involving different strains of Salmonella.     

Evolution of the tryptophan synthetase of fungi. Analysis of experimentally fused Escherichia coli tryptophan synthetase alpha and beta chains

During evolution of fungi, the separate tryptophan synthetase alpha and beta polypeptides of bacteria appear to have been fused in the order alpha-beta rather than the beta-alpha order that would be predicted from the order of the corresponding structural genes in all bacteria. We have fused the tryptophan synthetase polypeptides of Escherichia coli in both orders, alpha-beta and beta-alpha, with and without a short connecting (con) sequence, to explore possible explanations for the domain arrangement in fungi. We find that proteins composed of any of the four fused polypeptides, beta-alpha, beta-con-alpha, alpha-beta, and alpha-con-beta, are highly active enzymatically. However, only the alpha-beta and alpha-con-beta proteins are as active as the wild type enzyme. All four fusion proteins appear to be less soluble in vivo than the wild type enzyme; this abnormal characteristic is minimal for the alpha-con-beta enzyme. The alpha and beta domains of the four fusion polypeptides were not appreciably more heat labile than the wild type polypeptides. Competition experiments with mutant tryptophan synthetase alpha protein, and the fusion proteins suggest that in each fusion protein the joined alpha and beta domains have a functional tunnel connecting their alpha and beta active sites. Three tryptophan synthetase beta'-alpha fusion proteins were examined in which the carboxyl-terminal segment of the wild type beta polypeptide was deleted and replaced by a shorter, unnatural sequence. The resulting deletion fusion proteins were enzymatically inactive and were found predominantly in the cell debris. Evaluation of our findings in relation to the three-dimensional structure of the tryptophan synthetase enzyme complex of Salmonella typhimurium (5) and the results of mutational analyses with E. coli suggest that tryptophan synthetase may have evolved via an alpha-beta rather than a beta-alpha fusion because in beta-alpha fusions the amino-terminal helix of the alpha chain cannot assume the conformation required for optimal enzymatic activity.     

Repression of aromatic amino acid biosynthesis in Escherichia coli K-12

Mutants of Escherichia coli K-12 were isolated in which the synthesis of the following, normally repressible enzymes of aromatic biosynthesis was constitutive: 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetases (phe and tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A. In the wild type, DAHP synthetase (phe) was multivalently repressed by phenylalanine plus tryptophan, whereas DAHP synthetase (tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A were repressed by tyrosine. DAHP synthetase (tyr) and chorismate mutase T-prephenate dehydrogenase were also repressed by phenylalanine in high concentration (10(-3)m). Besides the constitutive synthesis of DAHP synthetase (phe), the mutants had the same phenotype as strains mutated in the tyrosine regulatory gene tyrR. The mutations causing this phenotype were cotransducible with trpA, trpE, cysB, and pyrF and mapped in the same region as tyrR at approximately 26 min on the chromosome. It is concluded that these mutations may be alleles of the tyrR gene and that synthesis of the enzymes listed above is controlled by this gene. Chorismate mutase P and prephenate dehydratase activities which are carried on a single protein were repressed by phenylalanine alone and were not controlled by tyrR. Formation of this protein is presumed to be controlled by a separate, unknown regulator gene. The heat-stable phenylalanine transaminase and two enzymes of the common aromatic pathway, 5-dehydroquinate synthetase and 5-dehydroquinase, were not repressible under the conditions studied and were not affected by tyrR. DAHP synthetase (trp) and tryptophan synthetase were repressed by tryptophan and have previously been shown to be under the control of the trpR regulatory gene. These enzymes also were unaffected by tyrR.     

tRNA(2Gln) mutants that translate the CGA arginine codon as glutamine in Escherichia coli.

We present a novel missense suppression system for the selection of tRNA(2GIn) mutants that can efficiently translate the CGA (arginine) codon as glutamine. tRNA(2Gln) mutants were cloned from a partially randomized synthetic gene pool using a plasmid vector that simultaneously expresses the tRNA gene and, to ensure efficient aminoacylation, the glutamine aminoacyl-tRNA synthetase gene (glnS). tRNA mutants that insert glutamine at CGA were selected as missense suppressors of a lacZ mutant (lacZ625(CGA)) that contains CGA substituted for an essential glutamine codon. Preliminary characterizations of four suppressors is presented. All of them contain two anticodon mutations: C-->U at position 34 and U-->C at position 35, which allow for cognate translation of CGA. U35 was previously shown to be an important determinant for glutaminylation of tRNA(2Gln) in vitro; suppression in vivo requires overexpression of the glutaminyl-tRNA synthetase gene (glnS). One tRNA variant contains no further mutations and has the highest missense suppression activity (8%). Three other isolates each contain an additional point mutation that alters suppression efficiency. This system will be useful for further studies of tRNA structure and function. In addition, because relatively efficient translation of the rare CGA codon as glutamine is not toxic for Escherichia coli, it may be possible to translate this sense codon with other alternate meanings, a property which could greatly facilitate protein engineering.