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C Yanofsky

Publications and source records attributed to C Yanofsky.

At least 19 recordsLinked to original sources
PubMed1995

TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, is a toroid-shaped molecule that binds transcripts containing GAG or UAG repeats separated by two nucleotides.

The trp RNA-binding attenuation protein of Bacillus subtilis, TRAP, regulates both transcription and translation by binding to specific transcript sequences. The optimal transcript sequences required for TRAP binding were determined by measuring complex formation between purified TRAP protein and synthetic RNAs. RNAs were tested that contained repeats of different trinucleotide sequences, with differing spacing between the repeats. A transcript containing GAG repeats separated by two-nucleotide spacers was bound most tightly. In addition, transmission electron microscopy was used to examine the structure of TRAP and the TRAP-transcript complex. TRAP was observed to be a toroid-shaped oligomer when free or when bound to either a natural or a synthetic RNA.

Bacillus subtilis↗
PubMed1995

Structural features of L-tryptophan required for activation of TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis.

A filter binding assay was used to determine the structural features of L-tryptophan required for activation of TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis. We examined the ability of L-tryptophan and 26 of its analogs to activate TRAP. Our findings show that TRAP activation by L-tryptophan is highly cooperative. We also observed that TRAP activation is stereospecific; D-tryptophan did not activate. Our results further indicate that the alpha-amino group and the carbonyl moiety of the alpha-carboxyl group of the ligand are required for TRAP activation and that the heterocyclic amino nitrogen of L-tryptophan greatly enhances TRAP activation. We also found that changes at several positions of the indole ring of L-tryptophan resulted in reduced TRAP activation. In addition, indole and 5-methylindole were shown to be effective competitors of L-tryptophan activation of TRAP.

Bacillus subtilis↗
PubMed1995

Light and developmental regulation of the gene con-10 of Neurospora crassa.

The gene con-10 of Neurospora crassa is expressed preferentially during conidiation and following illumination of vegetative mycelia with blue light. In this study we have examined the segmental locations of the genetic elements associated with con-10 that are responsible for light and developmental expression. A translational fusion was prepared between the initial segment of con-10 and Escherichia coli lacZ. Deletions were then introduced into the con-10 upstream region associated with this translational fusion. Each construct was integrated at the his-3 locus of N. crassa by transformation and homologous recombination. Photoinduction of mycelia containing the translational fusion with the intact upstream region revealed a two phase stimulus-response curve. Exposure to light for as little as 5 sec induced a transcriptional response. Following this initial induction, a period of 15 min in the dark or light was required for appearance of a second phase response. Only a brief light treatment was necessary for induction of the second phase response. Deletions within the upstream region altered normal light and developmental expression of constructs containing the con-10-lacZ translational fusion. The deleted segments appear to contain a mycelial repression site, two conidiation activation sites, and two dark repression sites. A repeated 17-bp sequence acted as a transcriptional enhancer. One copy of this enhancer is in the upstream region. The second copy, with the opposite orientation, is located in the first con-10 intron. The enhancer was required for proper mycelial and conidial expression of the con-10-lacZ fusion. The initial 10 bp of this enhancer sequence were sufficient to restore conidial expression to a deletion construct lacking both copies of the 17-bp repeat. Proteins were detected in extracts of mycelia and conidia that specifically bound to the enhancer sequence in vitro. Our findings suggest that conidiation-specific and mycelial-specific expression of con-10 requires the action of several factors acting independently and/or in concert at distinct sites located in the regulatory regions for con-10.

Base Sequence↗
PubMed1995

Bicyclomycin sensitivity and resistance affect Rho factor-mediated transcription termination in the tna operon of Escherichia coli.

The growth-inhibiting drug bicyclomycin, known to be an inhibitor of Rho factor activity in Escherichia coli, was shown to increase basal level expression of the tryptophanase (tna) operon and to allow growth of a tryptophan auxotroph on indole. The drug also relieved polarity in the trp operon and permitted growth of a trp double nonsense mutant on indole. Nine bicyclomycin-resistant mutants were isolated and partially characterized. Recombination data and genetic and biochemical complementation analyses suggest that five have mutations that affect rho, three have mutations that affect rpoB, and one has a mutation that affects a third locus, near rpoB. Individual mutants showed decreased, normal, or increased basal-level expression of the tna operon. All but one of the resistant mutants displayed greatly increased tna operon expression when grown in the presence of bicyclomycin. The tna operon of the wild-type drug-sensitive parent was also shown to be highly expressed during growth with noninhibitory concentrations of bicyclomycin. These findings demonstrate that resistance to this drug may be required by mutations at any one of three loci, two of which appear to be rho and rpoB.

Anti-Bacterial Agents↗
PubMed1995

trp RNA-binding attenuation protein (TRAP)-trp leader RNA interactions mediate translational as well as transcriptional regulation of the Bacillus subtilis trp operon.

Expression of the Bacillus subtilis trpEDCFBA operon has been shown to be regulated by transcription attenuation in response to the availability of L-tryptophan. Regulation is mediated by the tryptophan-activated trp RNA-binding attenuation protein, TRAP, the product of mtrB. Formation of mutually exclusive RNA anti-terminator and terminator structures within trp leader RNA determines whether transcription will terminate in the leader region of the operon. Previous studies suggested that transcripts that escape termination are subject to translational regulation via the formation of a secondary structure that blocks ribosome access to the trpE ribosome-binding site. To assess the relative importance of these postulated events in trp operon regulation, we used site-directed mutagenesis to alter the putative elements involved in transcriptional and translational control. Using a trpE'-'lacZ reporter, we measured translational yield and specific mRNA levels with various leader constructs, in both mtrB+ and mtrB strains, during growth in the presence and absence of excess tryptophan. To verify that the altered regulatory regions behaved as expected, we carried out in vitro transcription assays with the wild-type and altered leader region templates and performed oligonucleotide competition assays with an oligonucleotide complementary to a segment of the transcription terminator. Our results establish that binding of TRAP to leader RNA regulates of transcription termination in the trp operon over about an 88-fold range and regulates translation of trpE over about a 13-fold range. The roles played by different trp leader RNA segments in mediating transcriptional and translational regulation are documented by our findings.

Bacillus subtilis↗
PubMed1994

Mutants of Neurospora crassa that alter gene expression and conidia development.

Several genes have been identified that are highly expressed during conidiation. Inactivation of these genes has no observable phenotypic effect. Transcripts of two such genes, con-6 and con-10, are normally absent from vegetative mycelia. To identify regulatory genes that affect con-6 and/or con-10 expression, strains were prepared in which the regulatory regions for these genes were fused to a gene conferring hygromycin resistance. Mutants were then selected that were resistant to the drug during mycelial growth. Mutations in several of the isolates had trans effects; they activated transcription of the corresponding intact gene and, in most isolates, one or more of the other con genes. Most interestingly, resistant mutants were obtained that were defective at different stages of conidiation. One mutant conidiated under conditions that do not permit conidiation in wild type.

Cinnamates↗
PubMed1994

TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, is a multisubunit complex that appears to recognize G/UAG repeats in the trpEDCFBA and trpG transcripts.

A filter binding assay was developed to study interactions between purified TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, and trp specific transcripts. TRAP formed stable complexes with trpEDCFBA leader RNA; binding was L-tryptophan-dependent and was complete within 60 s. TRAP binds to a segment of the trp leader transcript that includes part of an RNA antiterminator structure. Binding to this segment allows formation of an RNA terminator structure, thereby promoting transcription termination. Using several trpEDCFBA leader deletion transcripts, we identified several closely spaced trinucleotide repeats (seven GAG and four UAG repeats) in the trp leader transcript that appeared to be required for TRAP binding. We also showed that TRAP binds to a segment of the trpG transcript that includes the trpG ribosome binding site; the nucleotide sequence of this segment contains several appropriately spaced trinucleotide repeats (seven GAG, one UAG, and one AAG). TRAP binding to the trpG transcript would block translation initiation. RNA footprint analysis confirmed interaction between TRAP and the trinucleotide repeats in the various transcripts. TRAP, in the presence or absence of L-tryptophan, appears to consist of 11 or 12 identical 8-kDa subunits. Our findings suggest that each tryptophan-activated TRAP subunit can bind one G/UAG repeat in a target transcript. Multiple protein-RNA interactions are required for stable association.

Bacillus subtilis↗
PubMed1994

Characterization of al-2, the phytoene synthase gene of Neurospora crassa. Cloning, sequence analysis, and photoregulation.

We have cloned the al-2 gene of Neurospora crassa and have analyzed its structure and regulation. The gene encodes a 603-residue polypeptide with a segment homologous to prokaryotic and other eukaryotic phytoene synthases. RNA measurements showed that the level of al-2 mRNA increased over 30-fold in photoinduced mycelia compared with dark-grown mycelia. This observation is consistent with the fact that carotenoid biosynthesis is induced by blue light during growth of N. crassa mycelia. The photoinduced increase in al-2 mRNA levels was not observed in two Neurospora mutants, wc-1 and wc-2, that are defective in all physiological photoresponses.

Alkyl and Aryl Transferases↗
PubMed1994

Mechanism of catabolite repression of tryptophanase synthesis in Escherichia coli.

Repression of tryptophanase (tryptophan indole-lyase) by glucose and its non-metabolizable analogue methyl alpha-glucoside has been studied employing a series of isogenic strains of Escherichia coli lacking cyclic AMP phosphodiesterase and altered for two of the proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), Enzyme I and Enzyme IIAGlc. Basal activity of tryptophanase was depressed mildly by inclusion of glucose in the growth medium, but inducible tryptophanase synthesis was subject to strong glucose repression in the parental strain, which exhibited normal PTS enzyme activities. Methyl alpha-glucoside was without effect in this strain. Loss of Enzyme I decreased sensitivity to repression by glucose but enhanced sensitivity to repression by methyl alpha-glucoside. Loss of Enzyme IIAGlc activity largely abolished repression by methyl alpha-glucoside but had a less severe effect on glucose repression. The repressive effects of both sugars were fully reversed by inclusion of cyclic AMP in the growth medium. Tryptophan uptake under the same conditions was inhibited weakly by glucose and more strongly by methyl alpha-glucoside in the parental strain. Inhibition by both sugars was alleviated by partial loss of Enzyme I. Inhibition by methyl alpha-glucoside appeared to be largely due to energy competition and was not responsible for repression of tryptophanase synthesis. Measurement of net production of cyclic AMP as well as intracellular concentrations of cyclic AMP revealed a good correlation with intensity of repression. The results suggest that while basal tryptophanase synthesis is relatively insensitive to catabolite repression, inducible synthesis is subject to strong repression by two distinct mechanisms, one dependent on enzyme IIAGlc of the PTS and the other independent of this protein. Both mechanisms are attributable to depressed rates of cyclic AMP synthesis. No evidence for a cyclic-AMP-independent mechanism of catabolite repression was obtained.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Role of regulatory features of the trp operon of Escherichia coli in mediating a response to a nutritional shift.

Physiological studies were performed under nutritional stress and nonstress conditions to assess the relative importance of the various regulatory mechanisms that Escherichia coli can use to alter its rate of tryptophan synthesis. Mutants were examined in which the trp repressor was inactive, transcription termination at the trp attenuator was altered, transcription initiation at the trp promoter was reduced, or feedback inhibition of anthranilate synthase was abolished. Strains were examined in media with and without tryptophan, phenylalanine and tyrosine, or acid-hydrolyzed casein and following shifts from one medium to another. Growth rates and anthranilate synthase levels were measured. In media lacking tryptophan, each of the mutants showed relief of repression and/or attenuation and maintained a near-normal growth rate. Following a shift from a medium containing tryptophan to a tryptophan-free medium containing phenylalanine and tyrosine or acid-hydrolyzed casein, mutants with abnormally low trp enzyme levels exhibited an appreciable growth lag before resuming growth. The wild-type strain displayed termination relief only under one extreme shift condition, upon transfer from a minimal medium containing tryptophan to minimal medium with only phenylalanine and tyrosine. A promoter down-mutant had difficulty adjusting to a shift from high tryptophan to low tryptophan levels in a medium containing acid-hydrolyzed casein. In all media tested, anthranilate synthase levels were lower in a feedback-resistant mutant than in the wild type. These studies demonstrate the capacity of E. coli to adjust its rate of tryptophan synthesis to maintain rapid growth following a shift to stressful nutritional conditions.

Adaptation, Biological↗
PubMed1993

Reconstitution of Bacillus subtilis trp attenuation in vitro with TRAP, the trp RNA-binding attenuation protein.

We have reconstituted Bacillus subtilis trp attenuation in vitro. Purification of the mtrB gene product (TRAP) to near homogeneity allowed us to demonstrate that addition of this protein plus L-tryptophan to template, RNA polymerase, and nucleoside triphosphates caused transcription termination in the trpEDCFBA leader region. TRAP acts by binding to the nascent transcript and preventing formation of an RNA antiterminator structure, thereby allowing terminator formation and transcription termination. Oligonucleotides complementary to segments of the antiterminator were used to demonstrate that formation of this RNA hairpin was responsible for transcription read-through. TRAP was found to be a 60-kDa multimeric protein composed of identical 6- to 8-kDa subunits, and its elution profile on a chromatographic column did not change in the presence of tryptophan.

Amino Acid Sequence↗
PubMed1992

Characterization of the tryptophanase operon of Proteus vulgaris. Cloning, nucleotide sequence, amino acid homology, and in vitro synthesis of the leader peptide and regulatory analysis.

The tryptophanase (tna) operon of Proteus vulgaris was cloned and characterized and found to be organized similarly to the tna operon of Escherichia coli. Both operons contain two major structural genes, tnaA and tnaB, that encode tryptophanase and a tryptophan permease, respectively. tnaA of P. vulgaris is preceded by a transcribed leader region, encoding a 34-residue leader peptide, TnaC, that contains a single tryptophan residue. The tnaC coding region also has a boxA-like sequence. Regulatory studies performed in P. vulgaris, and with a plasmid carrying the P. vulgaris tna operon in E. coli, established that expression of the Proteus operon was induced by tryptophan and was subject to catabolite repression. Site-directed mutagenesis studies established that translation of the tnaC coding region was essential for induction. Synthesis of the P. vulgaris leader peptide was demonstrated in an in vitro coupled transcription-translation system. Interestingly, the 5 amino acid residues of the TnaC peptide surrounding the sole tryptophan residue are identical in P. vulgaris and E. coli. We conclude that the tna operon of P. vulgaris is also regulated by tryptophan-induced transcription antitermination. Homology of tryptophanase and tryptophan permease of P. vulgaris to related proteins from other species is described.

Amino Acid Sequence↗
PubMed1992

trp repressor/trp operator interaction. Equilibrium and kinetic analysis of complex formation and stability.

The trp repressor of Escherichia coli regulates transcription initiation in the trp operon by binding at an operator located within the trp promoter region. We have used a filter binding assay to analyze the interaction between purified trp repressor and a synthetic 43-base pair DNA fragment containing the natural trp promoter-operator region. In equilibrium binding experiments, the KD of high affinity binding of trp repressor to this DNA fragment was determined to be 2 x 10(-10) M. Low affinity binding was observed at repressor concentrations above 10 nM. In kinetic experiments with various input ratios of repressor to operator, trp repressor-operator complexes dissociated with equivalent, first-order kinetics. Instantaneous reduction of the tryptophan concentration resulted in increased rates of complex dissociation, indicating that loss of one or both tryptophan molecules from the repressor-operator complex destabilizes the complex. A heterodimeric repressor with a single tryptophan binding site was constructed and its affinity for operator was compared with that of ligand free aporepressor and tryptophan saturated repressor. The heterodimeric repressor had a 20-25-fold higher affinity for operator than did the aporepressor, and it had a 20-25-fold lower affinity for operator than did the tryptophan-saturated repressor.

Bacterial Proteins↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

cot-1, a gene required for hyphal elongation in Neurospora crassa, encodes a protein kinase.

Neurospora crassa is a filamentous fungus that grows on semisolid media by forming spreading colonies. Mutations at several loci prevent this spreading growth. cot-1 is a temperature sensitive mutant of N.crassa that exhibits restricted colonial growth. At temperatures above 32 degrees C colonies are compact while at lower temperatures growth is indistinguishable from that of the wild type. Restricted colonial growth is due to a defect in hyphal tip elongation and a concomitant increase in hyphal branching. We have isolated a genomic cosmid clone containing the wild type allele of cot-1 by complementation. Sequence analyses suggested that cot-1 encodes a member of the cAMP-dependent protein kinase family. Strains in which we disrupted cot-1 are viable but display restricted colonial growth. Duplication, by ectopic integration of a promoter-containing fragment which includes the first one-third (209 codons) of the structural gene, unexpectedly resulted in restricted colonial growth. Our results suggest that an active COT1 kinase is required for one or more events essential for hyphal elongation.

Amino Acid Sequence↗
PubMed1992

A dominant selectable marker that is meiotically stable in Neurospora crassa: the amdS gene of Aspergillus nidulans.

When Neurospora crassa is transformed using a Neurospora gene as the selectable marker, the vegetatively stable transformants obtained cannot be used successfully in a cross because the selectable marker will be inactivated by the process of RIP (repeat-induced point mutation). Introduction of the acetamidase-encoding gene amdS of Aspergillus nidulans into N. crassa by transformation yielded transformants that would grow in minimal medium containing acetamide as a sole nitrogen source. In mitotically stable transformants containing a single copy of the amdS gene, the capacity to utilize acetamide as a sole nitrogen source was maintained in the progeny of a sexual cross. Therefore, the A. nidulans amdS gene is an appropriate dominant selectable marker for use in transformation analyses with N. crassa in which sexual crosses will be subsequently performed.

Amidohydrolases↗
PubMed1992

Timing of synthesis and cellular localization of two conidiation-specific proteins of Neurospora crassa.

The process of conidiation in Neurospora crassa consists of a series of distinct developmental stages culminating in the formation of multinucleate asexual spores called macroconidia. Immunoblotting techniques were used to study the timing of synthesis and cellular localization of CON10 and CON13, the products of two genes that are expressed during conidiation but not during mycelial growth. Both proteins first appear about 8 hr into conidiation; CON10 disappears between 2 and 4 hr after germination. Within conidiating cultures, CON10 and CON13 proteins are localized in conidiophores, with little or no protein present in the underlying mycelium. Immunofluorescence analyses show that CON10 is evenly distributed throughout the cytoplasm of macroconidia. Synthesis of CON10 and CON13 occurs at a time when their specifying mRNAs first appear (Hager and Yanofsky, Gene 96, 153-159, 1990; Sachs and Yanofsky, Dev. Biol 148, 117-128, 1991), suggesting that regulation of synthesis is predominantly transcriptional.

Antibodies↗