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C Yanofsky

Publications and source records attributed to C Yanofsky.

At least 19 recordsLinked to original sources
PubMed2005

New insights into regulation of the tryptophan biosynthetic operon in Gram-positive bacteria.

The tryptophan operon of Bacillus subtilis serves as an excellent model for investigating transcription regulation in Gram-positive bacteria. In this article, we extend this knowledge by analyzing the predicted regulatory regions in the trp operons of other fully sequenced Gram-positive bacteria. Interestingly, it appears that in eight of the organisms examined, transcription of the trp operon appears to be regulated by tandem T-box elements. These regulatory elements have recently been described in the trp operons of two bacterial species. Single T-box elements are commonly found in Gram-positive bacteria in operons encoding aminoacyl tRNA synthetases and proteins performing other functions. Different regulatory mechanisms appear to be associated with variations of trp gene organization within the trp operon.

Bacillus↗
PubMed2001

Inhibition of the B. subtilis regulatory protein TRAP by the TRAP-inhibitory protein, AT.

An anti-TRAP (AT) protein, a factor of previously unknown function, conveys the metabolic signal that the cellular transfer RNA for tryptophan (tRNATrp) is predominantly uncharged. Expression of the operon encoding AT is induced by uncharged tRNATrp. AT associates with TRAP, the trp operon attenuation protein, and inhibits its binding to its target RNA sequences. This relieves TRAP-mediated transcription termination and translation inhibition, increasing the rate of tryptophan biosynthesis. AT binds to TRAP primarily when it is in the tryptophan-activated state. The 53-residue AT polypeptide is homologous to the zinc-binding domain of DnaJ. The mechanisms regulating tryptophan biosynthesis in Bacillus subtilis differ from those used by Escherichia coli.

5' Untranslated Regions↗
PubMed2001

The mechanism of tryptophan induction of tryptophanase operon expression: tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNA(Pro).

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. In a previous study, we reproduced the regulatory features of this operon observed in vivo by using an in vitro S-30 system. We also found that, under inducing conditions, the leader peptidyl-tRNA (TnaC-peptidyl-tRNA(Pro)) is not cleaved; it accumulates in the S-30 reaction mixture. In this paper, we examine the requirements for TnaC-peptidyl-tRNA(Pro) accumulation and cleavage, in vitro. We show that this peptidyl-tRNA remains bound to the translating ribosome. Removal of free tryptophan and addition of release factor 1 or 2 leads to hydrolysis of TnaC-peptidyl-tRNA(Pro) and release of TnaC from the ribosome-mRNA complex. Release factor-mediated cleavage is prevented by the addition of tryptophan. TnaC of the ribosome-bound TnaC-peptidyl-tRNA(Pro) was transferable to puromycin. This transfer was also blocked by tryptophan. Tests with various tryptophan analogs as substitutes for tryptophan revealed the existence of strict structural requirements for tryptophan action. Our findings demonstrate that the addition of tryptophan to ribosomes bearing nascent TnaC-peptidyl-tRNA(Pro) inhibits both TnaC peptidyl-tRNA(Pro) hydrolysis and TnaC peptidyl transfer. The associated translating ribosome therefore remains attached to the leader transcript where it blocks Rho factor binding and subsequent transcription termination.

Bacterial Proteins↗
PubMed2001

The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, L-tryptophan.

The crystal structure of anthranilate synthase (AS) from Serratia marcescens, a mesophilic bacterium, has been solved in the presence of its substrates, chorismate and glutamine, and one product, glutamate, at 1.95 A, and with its bound feedback inhibitor, tryptophan, at 2.4 A. In comparison with the AS structure from the hyperthermophile Sulfolobus solfataricus, the S. marcescens structure shows similar subunit structures but a markedly different oligomeric organization. One crystal form of the S. marcescens enzyme displays a bound pyruvate as well as a putative anthranilate (the nitrogen group is ambiguous) in the TrpE subunit. It also confirms the presence of a covalently bound glutamyl thioester intermediate in the TrpG subunit. The tryptophan-bound form reveals that the inhibitor binds at a site distinct from that of the substrate, chorismate. Bound tryptophan appears to prevent chorismate binding by a demonstrable conformational effect, and the structure reveals how occupancy of only one of the two feedback inhibition sites can immobilize the catalytic activity of both TrpE subunits. The presence of effectors in the structure provides a view of the locations of some of the amino acid residues in the active sites. Our findings are discussed in terms of the previously described AS structure of S. solfataricus, mutational data obtained from enteric bacteria, and the enzyme's mechanism of action.

Amino Acid Sequence↗
PubMed2001

Advancing our knowledge in biochemistry, genetics, and microbiology through studies on tryptophan metabolism.

I was fortunate to practice science during the last half of the previous century, when many basic biological and biochemical concepts could be experimentally addressed for the first time. My introduction to research involved isolating and identifying intermediates in the niacin biosynthetic pathway. These studies were followed by investigations focused on determining the properties of genes and enzymes essential to metabolism and examining how they were alterable by mutation. The most challenging problem I initially attacked was establishing the colinear relationship between gene and protein. Subsequent research emphasized identification and characterization of regulatory mechanisms that microorganisms use to control gene expression. An elaborate regulatory strategy, transcription attenuation, was discovered that is often based on selection between alternative RNA structures. Throughout my career I enjoyed the excitement of solving basic scientific problems. Most rewarding, however, was the feeling that I was helping young scientists experience the pleasure of performing creative research.

Bacillus subtilis↗
PubMed2000

DNA microarray analysis of gene expression in response to physiological and genetic changes that affect tryptophan metabolism in Escherichia coli.

We investigated the global changes in mRNA abundance in Escherichia coli elicited by various perturbations of tryptophan metabolism. To do so we printed DNA microarrays containing 95% of all annotated E. coli ORFs. We determined the expression profile that is predominantly dictated by the activity of the tryptophan repressor. Only three operons, trp, mtr, and aroH, exhibited appreciable expression changes consistent with this profile. The quantitative changes we observed in mRNA levels for the five genes of the trp operon were consistent within a factor of 2, with expectations based on established Trp protein levels. Several operons known to be regulated by the TyrR protein, aroF-tyrA, aroL, aroP, and aroG, were down-regulated on addition of tryptophan. TyrR can be activated by any one of the three aromatic amino acids. Only one operon, tnaAB, was significantly activated by the presence of tryptophan in the medium. We uncovered a plethora of likely indirect effects of changes in tryptophan metabolism on intracellular mRNA pools, most prominent of which was the sensitivity of arginine biosynthetic operons to tryptophan starvation.

Escherichia coli↗
PubMed2000

Reproducing tna operon regulation in vitro in an S-30 system. Tryptophan induction inhibits cleavage of TnaC peptidyl-tRNA.

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. Catabolite repression regulates transcription initiation, whereas excess tryptophan induces antitermination at Rho factor-dependent termination sites in the leader region of the operon. Synthesis of the leader peptide, TnaC, is essential for antitermination. BoxA and rut sites in the immediate vicinity of the tnaC stop codon are required for termination. In this paper we use an in vitro S-30 cell-free system to analyze the features of tna operon regulation. We show that transcription initiation is cyclic AMP (cAMP)-dependent and is not influenced by tryptophan. Continuation of transcription beyond the leader region requires the presence of inducing levels of tryptophan and synthesis of the TnaC leader peptide. Using a tnaA'-'trpE fusion, we demonstrate that induction results in a 15-20-fold increase in synthesis of the tryptophan-free TnaA-TrpE fusion protein. Replacing Trp codon 12 of tnaC by an Arg codon, or changing the tnaC start codon to a stop codon, eliminates induction. Addition of bicyclomycin, a specific inhibitor of Rho factor action, substantially increases basal level expression. Analyses of tna mRNA synthesis in vitro demonstrate that, in the absence of inducer transcription is terminated and the terminated transcripts are degraded. In the presence of inducer, antitermination increases the synthesis of the read-through transcript. TnaC synthesis is observed in the cell-free system. However, in the presence of tryptophan, a peptidyl-tRNA also appears, TnaC-tRNA(Pro). Our findings suggest that inducer acts by preventing cleavage of TnaC peptidyl-tRNA. The ribosome associated with this newly synthesized peptidyl-tRNA presumably stalls at the tnaC stop codon, blocking Rho's access to the BoxA and rut sites, thereby preventing termination. 1-Methyltryptophan also is an effective inducer in vitro. This tryptophan analog is not incorporated into TnaC.

Bacterial Proteins↗
PubMed2000

A Bacillus subtilis operon containing genes of unknown function senses tRNATrp charging and regulates expression of the genes of tryptophan biosynthesis.

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.

Anthranilate Synthase↗
PubMed2000

Improving the catalytic activity of a thermophilic enzyme at low temperatures.

Enzymes from thermophilic organisms often are barely active at low temperatures. To obtain a better understanding of this sluggishness, we used DNA shuffling to mutagenize the trpC gene, which encodes indoleglycerol phosphate synthase, from the hyperthermophile Sulfolobus solfataricus. Mutants producing more active protein variants were selected by genetic complementation of an Escherichia coli mutant bearing a trpC deletion. Single amino acid changes and combinations of these changes improved growth appreciably. Five singly and doubly altered protein variants with changes at the N- and C-termini, or at the phosphate binding site, were purified and characterized with regard to their kinetics of enzymatic catalysis, product binding, cleavage by trypsin, and inactivation by heat. Turnover numbers of the purified variant proteins correlated with the corresponding growth rates, showing that the turnover number was the selected trait. Although the affinities for both the substrate and the product decreased appreciably in most protein variants, these defects were offset by the accumulation of high levels of the enzyme's substrate. Rapid mixing of the product indoleglycerol phosphate with the parental enzyme revealed that the enzyme's turnover number at low temperatures is limited by the dissociation of the enzyme-product complex. In contrast, representative protein variants bind and release the product far more rapidly, shifting the bottleneck to the preceding chemical step. The turnover number of the parental enzyme increases with temperature, suggesting that its structural rigidity is responsible for its poor catalytic activity at low temperatures. In support of this interpretation, the rate of trypsinolysis or of thermal denaturation is accelerated significantly in the activated protein variants.

Amino Acid Sequence↗
PubMed2000

Rho-dependent transcription termination in the tna operon of Escherichia coli: roles of the boxA sequence and the rut site.

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and by tryptophan-induced transcription antitermination. Tryptophan induction prevents Rho-dependent transcription termination in the leader region of the operon. Induction requires translation of a 24-residue leader peptide-coding region, tnaC, containing a single, crucial Trp codon. Studies with a lacZ reporter construct lacking the tnaC-tnaA spacer region suggest that, in the presence of excess tryptophan, the TnaC leader peptide acts in cis on the ribosome translating tnaC to inhibit its release. The stalled ribosome is thought to block Rho's access to the transcript. In this paper we examine the roles of the boxA sequence and the rut site in Rho-dependent termination. Deleting six nucleotides (CGC CCT) of boxA or introducing specific point mutations in boxA results in high-level constitutive expression. Some constitutive changes introduced in boxA do not change the TnaC peptide sequence. We confirm that deletion of the rut site results in constitutive expression. We also demonstrate that, in each constitutive construct, replacement of the tnaC start codon by a UAG stop codon reduces expression significantly, suggesting that constitutive expression requires translation of the tnaC coding sequence. Addition of bicyclomycin, an inhibitor of Rho, to these UAG constructs increases expression, demonstrating that reduced expression is due to Rho action. Combining a boxA point mutation with rut site deletion results in constitutive expression comparable to that of a maximally induced operon. These results support the hypothesis that in the presence of tryptophan the ribosome translating tnaC blocks Rho's access to the boxA and rut sites, thereby preventing transcription termination.

Amino Acid Sequence↗
PubMed2000

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

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.

Amino Acid Sequence↗
PubMed1999

Role of ribosome release in regulation of tna operon expression in Escherichia coli.

Expression of the degradative tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. In cultures growing in the absence of added tryptophan, transcription of the structural genes of the tna operon is limited by Rho-dependent transcription termination in the leader region of the operon. Tryptophan induction prevents this Rho-dependent termination, and requires in-frame translation of a 24-residue leader peptide coding region, tnaC, that contains a single, crucial, Trp codon. Studies with a lacZ reporter construct lacking the spacer region between tnaC and the first major structural gene, tnaA, suggested that tryptophan induction might involve cis action by the TnaC leader peptide on the ribosome translating the tnaC coding region. The leader peptide was hypothesized to inhibit ribosome release at the tnaC stop codon, thereby blocking Rho's access to the transcript. Regulatory studies with deletion constructs of the tna operon of Proteus vulgaris supported this interpretation. In the present study the putative role of the tnaC stop codon in tna operon regulation in E. coli was examined further by replacing the natural tnaC stop codon, UGA, with UAG or UAA in a tnaC-stop codon-tnaA'-'lacZ reporter construct. Basal level expression was reduced to 20 and 50% when the UGA stop codon was replaced by UAG or UAA, respectively, consistent with the finding that in E. coli translation terminates more efficiently at UAG and UAA than at UGA. Tryptophan induction was observed in strains with any of the stop codons. However, when UAG or UAA replaced UGA, the induced level of expression was also reduced to 15 and 50% of that obtained with UGA as the tnaC stop codon, respectively. Introduction of a mutant allele encoding a temperature-sensitive release factor 1, prfA1, increased basal level expression 60-fold when the tnaC stop codon was UAG and 3-fold when this stop codon was UAA; basal level expression was reduced by 50% in the construct with the natural stop codon, UGA. In strains with any of the three stop codons and the prfA1 mutation, the induced levels of tna operon expression were virtually identical. The effects of tnaC stop codon identity on expression were also examined in the absence of Rho action, using tnaC-stop codon-'lacZ constructs that lack the tnaC-tnaA spacer region. Expression was low in the absence of tnaC stop codon suppression. In most cases, tryptophan addition resulted in about 50% inhibition of expression when UGA was replaced by UAG or UAA and the appropriate suppressor was present. Introduction of the prfA1 mutant allele increased expression of the suppressed construct with the UAG stop codon; tryptophan addition also resulted in ca. 50% inhibition. These findings provide additional evidence implicating the behavior of the ribosome translating tnaC in the regulation of tna operon expression.

Bacterial Proteins↗
PubMed1997

Regulation of the Escherichia coli tna operon: nascent leader peptide control at the tnaC stop codon.

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and by tryptophan-induced transcription antitermination at Rho-dependent termination sites in the leader region of the operon. Tryptophan induction is dependent on translation of a short leader peptide coding region, tnaC, that contains a single, crucial tryptophan codon. Recent studies suggest that during induction, the TnaC leader peptide acts in cis on the translating ribosome to inhibit its release at the tnaC stop codon. In the present study we use a tnaC-UGA-'lacZ construct lacking the tnaC-tnaA spacer region to analyze the effect of TnaC synthesis on the behavior of the ribosome that translates tnaC. The tnaC-UGA-'lacZ construct is not expressed significantly in the presence or absence of inducer. However, it is expressed in the presence of UGA suppressors, or when the structural gene for polypeptide release factor 3 is disrupted, or when wild-type tRNATrP is overproduced. In each situation, tnaC-UGA-'lacZ expression is reduced appreciably by the presence of inducing levels of tryptophan. Replacing the tnaC UGA stop codon with a sense codon allows considerable expression, which is also reduced, although to a lesser extent, by the addition of tryptophan. Inhibition by tryptophan is not observed when Trp codon 12 of tnaC is changed to a Leu codon. Overexpression of tnaC in trans from a multicopy plasmid prevents inhibition of expression by tryptophan. These results support the hypothesis that the TnaC leader peptide acts in cis to alter the behavior of the translating ribosome.

Amino Acid Sequence↗
PubMed1997

Roles of the tnaC-tnaA spacer region and Rho factor in regulating expression of the tryptophanase operon of Proteus vulgaris.

To localize the DNA regions responsible for basal-level and induced expression of the tryptophanase (tna) operon of Proteus vulgaris, short deletions were introduced in the 115-bp spacer region separating tnaC, the leader peptide coding region, from tnaA. Deletions were incorporated into a tnaA'-'lacZ reporter construct containing the intact tna promoter-leader region. Expression was examined in Escherichia coli. Deletions that removed 28 to 30 bp from the region immediately following tnaC increased basal-level expression about threefold and allowed threefold induction by 1-methyltryptophan. A deletion removing 34 bp from the distal segment of the leader permitted basal and induced expression comparable to that of the parental construct. The mutant with the largest spacer deletion, 89 bp, exhibited a 30-fold increase in basal-level expression, and most importantly, inducer presence reduced operon expression by ca. 60%. Replacing the tnaC start codon or replacing or removing Trp codon 20 of tnaC of this deletion derivative eliminated inducer inhibition of expression. These findings suggest that the spacer region separating tnaC and tnaA is essential for Rho action. They also suggest that juxtaposition of the tnaC stop codon and the tnaA ribosome binding site in the 89-bp deletion derivative allows the ribosome that has completed translation of tnaC to inhibit translation initiation at the tnaA start codon when cells are exposed to inducer. These findings are consistent with results in the companion article that suggest that inducer allows the TnaC peptide to inhibit ribosome release at the tnaC stop codon.

Bacterial Proteins↗
PubMed1996

On the role of helix 0 of the tryptophan synthetase alpha chain of Escherichia coli.

The role of helix 0 of the alpha chain (TrpA) of the tryptophan synthetase alpha2beta2 multi-functional enzyme complex of Escherichia coli was examined by deleting amino-terminal residues 2-6, 2-11, or 2-19 of TrpA. Selected substitutions were also introduced at TrpA positions 2-6. The altered genes encoding these polypeptides were overexpressed from a foreign promoter on a multicopy plasmid and following insertion at their normal chromosomal location. Each deletion polypeptide was functional in vivo. However all appeared to be somewhat more labile and insoluble and less active enzymatically than wild type TrpA. The deletion polypeptides were overproduced and solubilized from cell debris by denaturation and refolding. Several were partially purified and assayed in various reactions in the presence of tryptophan synthetase beta2 (TrpB). The purified TrpADelta2-6 and TrpADelta2-11 deletion polypeptides had low activity in both the indole + serine --> tryptophan reaction and the indoleglycerol phosphate + serine --> tryptophan reaction. Poor activity in each reaction was partly due to reduced association of TrpA with TrpB. The addition of the TrpA ligands, alpha-glycerophosphate or indoleglycerol phosphate, during catalysis of the indole + serine --> tryptophan reaction increased association and activity. These findings suggest that removal of helix 0 of TrpA decreases TrpA-TrpB association as well as the activity of the TrpA active site. Alignment of the TrpA sequences from different species indicates that several lack part or all of helix 0. In some of these polypeptides, extra residues at the carboxyl end may substitute for helix 0.

Amino Acid Sequence↗
PubMed1996

Flexibility of DNA binding domain of trp repressor required for recognition of different operator sequences.

Trp repressor (25 kDa) is a regulatory protein that controls transcription initiation in the tryptophan biosynthetic operon and at least four other operons in Escherichia coli. An alanine to valine mutation (AV77) in the DNA binding domain is known to increase repressor activity at the trp operator in vivo, but not in vitro. We report here the amide proton exchange rates for the DNA-binding domains of both the wild-type and AV77 proteins. We find that the alanine to valine change stabilizes the flexible DNA-binding domain of the repressor. We present in vivo data showing that, although the AV77 repressor is more inhibitory at the trp operator than the wild-type repressor, it does not have increased activity at the aroH or trpR operator; repression at the aroH operator is, in fact, reduced. Our results suggest that the flexibility exhibited by the wild-type repressor allows a broader range of repressor/DNA interactions, whereas the increased rigidity resulting from the AV77 change limits the repressor's effectiveness at some operators.

Amides↗
PubMed1996

Some novel transcription attenuation mechanisms used by bacteria.

A variety of transcription attenuation mechanisms are used by bacteria to regulate gene and operon expression. This review summarizes previous and current studies designed to elucidate the features of the specific attenuation mechanisms that regulate expression of the tryptophanase (tna) operon of Escherichia coli and the tryptophan (trp) operon of Bacillus subtilis. Initiation of transcription in the tna operon is regulated by catabolite repression. Once initiated, transcription is regulated by tryptophan-induced inhibition of Rho-mediated transcription termination in the leader region of the operon. An operon-encoded leader peptide, TnaC, containing a crucial tryptophan residue, plays an essential role in induction. This peptide appears to act in cis on the ribosome translating tnaC to inhibit its release at the tnaC stop codon. The stalled ribosome would block Rho's access to the tna transcript, thereby preventing termination. Transcription of the trp operon of B subtilis is regulated by an attenuation mechanism that responds to a tryptophan-activated eleven subunit RNA-binding regulatory protein, called TRAP. Activated TRAP binds to repeated GAG sequences in the leader segment of the trp operon transcript, disrupting an RNA antiterminator and promoting formation of a terminator. Activated TRAP also regulates translation of trpG in the folate operon by binding to repeat GAG sequences surrounding the trpG ribosome binding site. A temperature sensitive tryptophanyl-tRNA synthetase (trpS) mutant was previously observed to overexpress the trp operon and trpG, when grown at elevated temperatures in the presence of tryptophan. We have found that the trpS defect increases trp operon and trpG expression by interfering with TRAP's ability to act. We suggest that either accumulation of uncharged tRNA(Trp) or overproduction of a TRAP-binding transcript reduces the level of functional TRAP in the trpS mutant.

Amino Acid Sequence↗