An alkaline phosphatase protection assay to investigate trp repressor/operator interactions

We have used an alkaline phosphatase protection assay to investigate the interaction of the trp repressor with its operator sequence. The assay is based on the principle that the trp repressor will protect a terminally 5'-32P-labeled operator DNA fragment from attack by alkaline phosphatase. The optimal oligonucleotide for investigating the trp repressor/operator interaction extends two base pairs from each end of the genetically defined target sequence predicted by in vivo studies [Bass et al. (1987) Genes Dev. 1, 565-572]. The assay works well over a 10,000-fold range of protein/DNA affinity and is used to show that the corepressor, L-tryptophan, causes the liganded repressor to bind a 20 base pair trp operator duplex 6400 times more strongly than the unliganded aporepressor. The affinity of the trp repressor for operators containing symmetrical mutations was interpreted in terms of the trp repressor/operator crystal structure as follows: (1) Direct hydrogen bonds with the functional groups of G-9 of the trp operator and the side chain of Arg 69 of the trp repressor contribute to DNA-binding specificity. (2) G-6 of the trp operator is critical for DNA-binding specificity probably because of the two water-mediated hydrogen bonds between its functional groups and the N-terminus of the trp repressor's E-helix. (3) Sequence-dependent aspects of the trp operator's conformation help stabilize the trp repressor/operator complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppressors of trp1 fluorescence identify a new arabidopsis gene, TRP4, encoding the anthranilate synthase beta subunit.

Suppressors of the blue fluorescence phenotype of the Arabidopsis trp1-100 mutant can be used to identify mutations in genes involved in plant tryptophan biosynthesis. Two recessive suppressor mutations define a new gene, TRP4. The trp4 mutant and the trp1-100 mutant are morphologically normal and grow without tryptophan, whereas the trp4; trp1-100 double mutant requires tryptophan for growth. The trp4; trp1-100 double mutant does not segregate at expected frequencies in genetic crosses because of a female-specific defect in transmission of the double mutant genotype, suggesting a role for the tryptophan pathway in female gametophyte development. Genetic and biochemical evidence shows that trp4 mutants are defective in a gene encoding the beta subunit of anthranilate synthase (AS). Arabidopsis AS beta subunit genes were isolated by complementation of an Escherichia coli anthranilate synthase mutation. The trp4 mutation cosegregates with one of the genes, ASB1, located on chromosome 1. Sequence analysis of the ASB1 gene from trp4-1 and trp4-2 plants revealed different single base pair substitutions relative to the wild type. Anthranilate synthase alpha and beta subunit genes are regulated coordinately in response to bacterial pathogen infiltration.     

Suppression of Two Missense Alleles of the TRP5 Locus of SACCHAROMYCES CEREVISIAE

A suppressor SUP101 of alleles trp5-67 and trp5-18 of the trp5 locus of Saccharomyces cerevisiae is described. The two suppressible mutations have been previously classified as missense. The suppression does not result from a physiological bypass of the tryptophan synthetase-catalyzed reaction, since the suppression is allele-specific. IU alleles trp5-70, tryp5-95, and trp5-102; IA alleles trp5-81, trp5-101, and trp5-103; and the ochre alleles trp5-33 and trp5-48 are not suppressed by SUP101. SUP101 does not suppress ochre alleles ade2-1, his5-2, arg4-17, lys1-1, amber alleles trp1-1, tyr7-1, or unclassified alleles at a number of other loci. These results indicate SUP101 is a missense suppressor. Growth on tryptophanless media is dependent upon gene dosage of both the suppressor and the suppressible alleles. Only the diploids homozygous both for the suppressor and suppressible alleles produce growth equivalent to growth of the haploids bearing a suppressible allele and the suppressor. Suppressor-bearing strains grow poorly even on tryptophan-supplemented media. In more than 100 asci analyzed partial growth inhibition on the complete medium always segregated with the suppressor.     

5-Fluoroindole Resistance Identifies Tryptophan Synthase Beta Subunit Mutants in Arabidopsis Thaliana

A study of the biochemical genetics of the Arabidopsis thaliana tryptophan synthase beta subunit was initiated by characterization of mutants resistant to the inhibitor 5-fluoroindole. Thirteen recessive mutations were recovered that are allelic to trp2-1, a mutation in the more highly expressed of duplicate tryptophan synthase beta subunit genes (TSB1). Ten of these mutations (trp2-2 through trp2-11) cause a tryptophan requirement (auxotrophs), whereas three (trp2-100 through trp2-102) remain tryptophan prototrophs. The mutations cause a variety of changes in tryptophan synthase beta expression. For example, two mutations (trp2-5 and trp2-8) cause dramatically reduced accumulation of TSB mRNA and immunologically detectable protein, whereas trp2-10 is associated with increased mRNA and protein. A correlation exists between the quantity of mutant beta and wild-type alpha subunit levels in the trp2 mutant plants, suggesting that the synthesis of these proteins is coordinated or that the quantity or structure of the beta subunit influences the stability of the alpha protein. The level of immunologically detectable anthranilate synthase alpha subunit protein is increased in the trp2 mutants, suggesting the possibility of regulation of anthranilate synthase levels in response to tryptophan limitation.     

Influence of Chromosome Structure on the Frequency of tonB trp Deletions in Escherichia coli

The frequency of tonB trp deletions varies in different strains and substrains of Escherichia coli. Studies with chromosomal hybrids constructed by transducing various segments of the cysB-trp-suIII region from K-12(Ymel) into K-12(W3110) indicate that the characteristic low deletion frequency of K-12(Ymel) is determined largely by the (genetic) structure of the trp-suIII region of the chromosome. Transduction of the trp region from K-12(W3110) or K-12(Ymel) into strain B has little effect on the frequency of tonB trp deletions in that strain. When tonB trp deletions occur at 42 C rather than at 37 C, there is a significant reduction in the frequency of deletions in all strains examined except K-12(Ymel) and hybrids exhibiting a Ymel deletion pattern. The magnitude of this temperature effect in different K-12 strains increases proportionally with the frequency of tonB trp deletions at 37 C. At 42 C the frequency of tonB trp deletions in all K-12 strains approaches the low frequency observed for Ymel at 37 or 42 C. In contrast, spontaneous deletions in another region of the genome which simultaneously result in resistance to phages T7 and lambda and in proline auxotrophy (tfrA pro deletions) occur at a constant frequency regardless of growth temperature or the structure of the chromosome in the trp region. Two mutants of strain KB30 obtained after treatment with nitrosoguanidine show very low tonB trp deletion frequencies. The alterations in both mutants map in the trp region of the chromosome. These studies indicate that the structure of the cysB-trp-suIII region is responsible for many of the characteristic deletion frequencies observed.     

Isolation and Characterization of Specialized φ80 Transducing Phages Carrying Regions of the Salmonella typhimurium trp Operon

We have isolated a series of nondefective phi80 specialized transducing phage which carry segments of the Salmonella typhimurium trp operon. These phage were obtained from a lysogenic derivative of a merozygote constructed by transferring an S. typhimurium trp episome into an Escherichia coli strain which lacks the normal phi80 attachment site. The deoxyribonucleic acid (DNA) from one such phage was purified and employed in DNA-ribonucleic acid (RNA) hybridization studies. The results obtained show that, under our hybridization conditions, heterologous hybridization is less efficient than homologous hybridization. It was also observed that not all S. typhimurium trp messenger RNA can readily anneal to E. coli trp operon DNA. Heterologous hybrids consisting of S. typhimurium trp messenger RNA and E. coli trp operon DNA were estimated to have a dissociation constant 10-fold larger than that of homologous hybrids.     

The NH2-terminal arms of trp repressor participate in repressor/operator association.

The 3-dimensional structure of the trp repressor, aporepressor, and repressor/operator complex have been described. The NH2-terminal arms of the protein, comprising approximately 12-14 residues, were not well resolved in any of these structures. Previous studies by Carey showed that the arms are required for full in vitro repressor activity. To examine the roles of the arms more fully we have removed codons 2-5 and 2-8 of the trpR gene and analyzed the resulting truncated repressors in vivo and in vitro. The delta 2-5 trp repressor was found to be approximately 25% as active as the wild type repressor in vivo. In in vitro equilibrium binding experiments, the delta 2-5 trp repressor was shown to be five-fold less active in operator binding. The rate of dissociation of the complex formed between the delta 2-5 trp repressor and operator was essentially the same as the rate of dissociation of the wild type trp repressor/operator complex. However association of the delta 2-5 trp repressor with operator was clearly defective. Since the NH2-terminal arms of the trp repressor appear to affect association predominantly they may play a role in facilitating non-specific association of repressor with DNA as repressor seeks its cognate operators. The delta 2-8 trp repressor was unstable in vivo and in vitro, suggesting that some portion of the NH2-terminal arm is required for proper folding of the remainder of the molecule.     

Iron-related modification of bacterial transfer RNA

Transfer RNAs isolated from E. coli grown in media where ferric iron is not freely available show well characterized chromatographic changes due to the absence of the methylthio moiety of ms2i6A. The altered tRNA molecules include tRNA trp tRNA tyr, tRNA phe and two minor tRNA ser species. It has been suggested that methylthiolation of tRNA affects its function in regulation. We now show iron-related changes in tRNA trp from S. typhimurium, Ps. aeruginosa and K. pneumoniae. tRNA trp from S. typhimurium contains ms2i6A and it seems probable that the availability of iron affects the synthesis of ms2i6A-tRNA trp from i6A-tRNA trp in this organism. An iron-related methylthiolating system may also be operative in K. pneumoniae. S. marcescens tRNA trp, however was not affected by the availability of iron. Neither ms2i6A nor i6A was found in S. marcescens tRNA, although an, as yet unidentified, hydrophobic nucleoside was present.     

Effect of tryptophan analogs on derepression of the Escherichia coli tryptophan operon by indole-3-propionic acid.

The abilities of 14 tryptophan analogs to repress the tryptophan (trp) operon have been studied in Escherichia coli cells derepressed by incubation with 0.25 mM indole-3-propionic acid (IPA). trp operon expression was monitored by measuring the specific activities of anthranilate synthase (EC 4.1.3.27) and the tryptophan synthase (EC 4.2.1.20) beta subunit. Analogs characterized by modification or removal of the alpha-amino group or the alpha-carboxyl group did not repress the trp operon. The only analogs among this group that appeared to interact with the trp aporepressor were IPA, which derepressed the trp operon, and d-tryptophan. Analogs with modifications of the indole ring repressed the trp operon to various degrees. 7-Methyl-tryptophan inhibited anthranilate synthase activity and consequently derepressed the trp operon. Additionally, 7-methyltryptophan prevented IPA-mediated derepression but, unlike tryptophan, did so in a non-coordinate manner, with the later enzymes of the operon being relatively more repressed than the early enzymes. The effect of 7-methyltryptophan on IPA-mediated derepression was likely not due to the interaction of IPA with the allosteric site of anthranilate synthase, even though feedback-resistant mutants of anthranilate synthase were partially resistant to derepression by IPA. The effect of 7-methyltryptophan on derepression by IPA was probably due to the effect of the analog-aporepressor complex on trp operon expression.     

Isolation of specialized transducing bacteriophages carrying deletions of the regulatory region of the Escherichia coli K-12 tryptophan operon.

A lysogen of a wild-type strain of Escherichia coli K-12 carrying a heat-inducible lambda-phi80 hybrid prophage was induced to yield transducing phages carrying all of the structural genes of the tryptophan operon. The presence or absence of elements of the trp regulatory region was determined by examining the effects of lambda genes N and cI on trp gene expression. The phages were further characterized by transduction studies and by examining anthranilate synthetase (EC 4.1.3.27) (TRYPE+D) synthesis in the presence of the lambda cI product. A number of phages deleted for the trp promoter were found. Recombination studies between trpOc bacteria and the transducing phages have yielded information that can be used to order the trp end points of some phages and to provide an estimate of the size of the trp promoter region.