Deletion plasmids from transformants of Pseudomonas aeruginosa trp cells with the RSF1010-trp hybrid plasmid and high levels of enzyme activity from the gene on the plasmid. (1/162)

A RSF1010-trp hybrid plasmid which contained the tryptophan operon of Escherichia coli was introduced into Pseudomonas aeruginosa trp cells by transformation. From the Trp+ transformants several deletion plasmids were obtained, and their physical maps with restriction endonucleases were constructed. P. aeruginosa trp cells with these plasmids showed at first more than 100 times higher levels of tryptophan synthetase beta activity over that of the control P. aeruginosa wild-type cells, but these levels were drastically decreased by 1 week of successive transfers of cultures. This decrease in enzyme activity was found to be due to the change on the plasmids but not to the host cells. The production of E. coli tryptophan synthetase beta enzyme in P. aeruginosa cells was proved by immunological test.  (+info)

The crystal structure of anthranilate synthase from Sulfolobus solfataricus: functional implications. (2/162)

Anthranilate synthase catalyzes the synthesis of anthranilate from chorismate and glutamine and is feedback-inhibited by tryptophan. The enzyme of the hyperthermophile Sulfolobus solfataricus has been crystallized in the absence of physiological ligands, and its three-dimensional structure has been determined at 2.5-A resolution with x-ray crystallography. It is a heterotetramer of anthranilate synthase (TrpE) and glutamine amidotransferase (TrpG) subunits, in which two TrpG:TrpE protomers associate mainly via the TrpG subunits. The small TrpG subunit (195 residues) has the known "triad" glutamine amidotransferase fold. The large TrpE subunit (421 residues) has a novel fold. It displays a cleft between two domains, the tips of which contact the TrpG subunit across its active site. Clusters of catalytically essential residues are located inside the cleft, spatially separated from clustered residues involved in feedback inhibition. The structure suggests a model in which chorismate binding triggers a relative movement of the two domain tips of the TrpE subunit, activating the TrpG subunit and creating a channel for passage of ammonia toward the active site of the TrpE subunit. Tryptophan presumably blocks this rearrangement, thus stabilizing the inactive states of both subunits. The structure of the TrpE subunit is a likely prototype for the related enzymes 4-amino 4-deoxychorismate synthase and isochorismate synthase.  (+info)

Inhibition of TATA-binding protein function by SAGA subunits Spt3 and Spt8 at Gcn4-activated promoters. (3/162)

SAGA is a 1.8-MDa yeast protein complex that is composed of several distinct classes of transcription-related factors, including the adaptor/acetyltransferase Gcn5, Spt proteins, and a subset of TBP-associated factors. Our results indicate that mutations that completely disrupt SAGA (deletions of SPT7 or SPT20) strongly reduce transcriptional activation at the HIS3 and TRP3 genes and that Gcn5 is required for normal HIS3 transcriptional start site selection. Surprisingly, mutations in Spt proteins involved in the SAGA-TBP interaction (Spt3 and Spt8) cause derepression of HIS3 and TRP3 transcription in the uninduced state. Consistent with this finding, wild-type SAGA inhibits TBP binding to the HIS3 promoter in vitro, while SAGA lacking Spt3 or Spt8 is not inhibitory. We detected two distinct forms of SAGA in cell extracts and, strikingly, one lacks Spt8. Conditions that induce HIS3 and TRP3 transcription result in an altered balance between these complexes strongly in favor of the form without Spt8. These results suggest that the composition of SAGA may be dynamic in vivo and may be regulated through dissociable inhibitory subunits.  (+info)

A Bacillus subtilis operon containing genes of unknown function senses tRNATrp charging and regulates expression of the genes of tryptophan biosynthesis. (4/162)

Strains of Bacillus subtilis containing a temperature-sensitive tryptophanyl-tRNA synthetase produce elevated levels of the tryptophan pathway enzymes, when grown at high temperatures in the presence of excess tryptophan. This increase is because of reduced availability of the tryptophan-activated trp RNA-binding attenuation protein (TRAP). To test the hypothesis that this elevated trp gene expression was caused by the overproduction of a transcript capable of binding and sequestering TRAP, a computer program was designed to search the B. subtilis genome sequence for additional potential TRAP binding sites. A region containing a stretch of (G/A)AG trinucleotide repeats, characteristic of a TRAP binding site, was identified in the yczA-ycbK operon. We show that transcriptional regulation of the yczA-ycbK operon is controlled by the T-box antitermination mechanism in response to the level of uncharged tRNA(Trp), and that the presence of a trpS1 mutant allele increases production of the yczA-ycbK transcript. Elevated yczA-ycbK expression was shown to activate transcription of the trp operon. Deletion of the yczA-ycbK operon abolishes the trpS1 effect on trp gene expression. The purpose of increasing expression of the genes of tryptophan biosynthesis in the trpS mutant would be to provide additional tryptophan to overcome the charged tRNA(Trp) deficiency. Therefore, in B. subtilis, as in Escherichia coli, transcription of the tryptophan biosynthetic genes is regulated in response to changes in the extent of charging of tRNA(Trp) as well as the availability of tryptophan.  (+info)

A Bacillus subtilis gene of previously unknown function, yhaG, is translationally regulated by tryptophan-activated TRAP and appears to be involved in tryptophan transport. (5/162)

Computer analysis of the Bacillus subtilis genome sequence revealed a gene with no previously attributed function, yhaG, specifying a transcript containing a presumptive binding site for the tryptophan-activated regulatory protein, TRAP. The presumptive TRAP binding site overlaps the yhaG Shine-Dalgarno sequence and translation initiation region. TRAP was shown to regulate expression of yhaG translationally. Production of the yhaG transcript in vivo was found to compete for the binding of TRAP to other known TRAP binding sites. YhaG is likely to be a transmembrane protein involved in tryptophan transport.  (+info)

The nucleotide sequence of the first externally suppressible--1 frameshift mutant, and of some nearby leaky frameshift mutants. (6/162)

Nine mutants within a 23 nucleotide sequence of the trpE gene of Salmonella typhimurium have been characterized. trpE91, a mutant which is externally suppressible has a single base deletion. Eight (or nine) nucleotides upstream of this deletion, two independently isolated mutations have the same transversion. In combination with trpE91 these mutations lead to partial restoration of synthesis of anthranilate synthetase in the absence of external suppressors. In the transversion the sequence A CA is changed to A AA and this new sequence may be the site where frameshifting occurs to allow leakiness. Leakiness is displayed by two further mutants of the same sign as trpE91, and one of the opposite sign, in the absence of any base substitution or external suppressors. Specific sequences, e.g., UUUC, may be especially prone to frameshifting and this sequence is created at the site of the +1 frameshift mutant which displays leakiness. In the new reading frame generated by the two -1 frame leaky mutants, a tryptophan codon is encountered. Leakiness is necessarily detected in the absence of tryptophan and under these conditions there will be a shortage of charged tryptophan tRNA. The possibility of such functional imbalance leading to frameshifting in these mutants is discussed.  (+info)

Increasing tryptophan synthesis in a forage legume Astragalus sinicus by expressing the tobacco feedback-insensitive anthranilate synthase (ASA2) gene. (7/162)

A cDNA clone that encodes a feedback-insensitive anthranilate synthase (AS), ASA2, isolated from a 5-methyl-tryptophan (Trp) (5MT)-resistant tobacco cell line under the control of the constitutive cauliflower mosaic virus 35S promoter, was introduced into the forage legume Astragalus sinicus by Agrobacterium rhizogenes with kanamycin selection. The 35S-ASA2 gene was expressed constitutively as demonstrated by northern-blot hybridization analyses and the presence of feedback-insensitive AS. Hairy root lines transformed with 35S-ASA2 grew in concentrations of up to 100 microM 5MT, whereas the controls were completely inhibited by 15 microM 5MT. Expression of the feedback-insensitive ASA2 resulted in a 1.3- to 5.5-fold increase in free Trp. Kinetic studies of the AS activity demonstrate the Trp feedback alterations and indicate that the ASA2 alpha-subunit can interact with the native A. sinicus beta-subunit to form an active enzyme. The ASA2 transcript and high free Trp were also detected in the leaves, stems, and roots of plants regenerated from the transformed hairy roots. Thus, we show for the first time that ASA2 can be used to transform plants of a different species to increase the levels of the essential amino acid Trp and impart 5MT resistance.  (+info)

Decay of mutualistic potential in aphid endosymbionts through silencing of biosynthetic loci: Buchnera of Diuraphis. (8/162)

Buchnera, the primary bacterial endosymbiont of aphids, is known to provision essential amino acids lacking in the hosts' diet of plant sap. The recent discovery of silenced copies of genes for tryptophan biosynthesis (trpEG) in certain Buchnera lineages suggests a decay in symbiotic functions in some aphid species. However, neither the distribution of pseudogenes among lineages nor the impact of this gene silencing on amino-acid availability in hosts has been assessed. In Buchnera of the aphid Diuraphis noxia, tandem repeats of these pseudogenes have persisted in diverse lineages, and thpEG pseudogenes have originated at least twice within this aphid genus. Measures of amino-acid concentrations in Diuraphis species have shown that the presence of the pseudogene is associated with a decreased availability of tryptophan, indicating that gene silencing decreases nutrient provisioning by symbionts. In Buchnera of Diuraphis, rates of nonsynonymous substitutions are elevated in functional trpE copies, supporting the hypothesis that pseudogene origin and persistence reflect a reduced selection for symbiont biosynthetic contributions. The parallel evolution of trpEG pseudogenes in Buchnera of Diuraphis and certain other aphid hosts suggests that either selection at the host level is not effective or that fitness in these aphids is not limited by tryptophan availability.  (+info)

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