Biomedical subjects

C Yanofsky

Publications and source records attributed to C Yanofsky.

At least 127 records · Page 7Linked to original sources

PubMed1985

Translation activates the paused transcription complex and restores transcription of the trp operon leader region.

It has been proposed that RNA polymerase pausing in the leader region of the tryptophan (trp) operon of Escherichia coli is responsible for the synchronization of transcription and translation essential to attenuation control. In this report we use an in vitro coupled transcription/translation system to study the effect of trp leader peptide synthesis on RNA polymerase pausing in the trp leader region. Wild-type and translation-defective trp leader templates of E. coli and Serratia marcescens were employed, and pause RNA synthesis and paused complex release (activation) were quantified relative to synthesis of the terminated leader transcript. It was observed that pausing in the trp leader region was prolonged when translation of the leader transcript was reduced by mutations in the leader region or by addition of the translation inhibitor kasugamycin or chloramphenicol. Experiments with S-30 extracts from a mutant strain that is inefficient in translating the tryptophan codons in the leader transcript indicated that ribosome movement to these codons also releases the paused transcription complex. These findings indicate that the paused trp leader transcription complex resumes transcription when released by ribosome movement over the leader peptide coding region. This release would facilitate the coupling of transcription and translation essential to attenuation control.

Amino Acid Sequence↗

PubMed1985

Mutational studies with the trp repressor of Escherichia coli support the helix-turn-helix model of repressor recognition of operator DNA.

Several classes of trp repressor mutants were selected and analyzed in vivo. Mutants that produced repressors with either enhanced or reduced activity were obtained. One class of mutants produced inactive or slightly active repressors that were trans-dominant to the wild-type repressor. The amino acid substitutions in many of these repressors were clustered in a segment of the polypeptide that is homologous to the DNA recognition domain of the lambda cro repressor. A second functionally important region of the trp repressor was identified; this region could participate in L-tryptophan binding. Observations with trpR nonsense mutants suggest that the first 67 residues of the repressor polypeptide are sufficient for subunit association.

Amino Acid Sequence↗

PubMed1985

Evidence for transcription antitermination control of tryptophanase operon expression in Escherichia coli K-12.

Tryptophanase, encoded by the gene tnaA, is a catabolic enzyme distinct from the enzymes of tryptophan biosynthesis. Tryptophanase synthesis is induced by tryptophan and is subject to catabolite repression. We studied the mechanism of tna operon induction. Mutants with altered rho factor were partially constitutive for tna expression, implicating rho-dependent transcription termination in the control of tna expression. Measurements of mRNA synthesis from the transcribed leader region preceeding the tna operon suggested that the tna promoter was constitutive and that in the absence of inducer, transcription terminated in the leader region. Upon induction, this transcription termination was relieved. Cis-acting constitutive mutants had genetic alterations in the tna leader region. These lesions defined a site that is homologous to the bacteriophage lambda boxA sequence, which is thought to play a role in antitermination control of lambda lytic gene expression. We propose that tna expression is subject to transcription antitermination control. We hypothesize that a tryptophan-activated antiterminator protein mediates induction by suppressing the rho-dependent termination sites in the leader region, thus allowing transcription to proceed into the tna operon structural gene region.

Base Sequence↗

PubMed1985

Protein changes during the asexual cycle of Neurospora crassa.

A method for synchronizing conidiation and isolating large numbers of cells at discrete stages of conidia development is described. Using two-dimensional gel electrophoresis, we analyzed the protein profiles of mycelia, aerial hyphae, and conidia and observed that the concentration of 14 polypeptides increase and 38 decrease during the asexual cycle. Twelve polypeptides were present in extracts of aerial hyphae or conidia, but not mycelia, suggesting that they may be conidiation specific. The protein profiles of mutants defective in conidiation were also analyzed. Differences were detected in the two-dimensional profiles of protein extracts from fluffy and wild-type aerial hyphae. Polyadenylated RNA isolated from wild-type mycelia and conidiating cultures was translated in vitro in a rabbit reticulocyte lysate. Differences were detected in the polypeptide products specified by the two RNA populations, suggesting that changes in steady-state levels of polyadenylated RNAs also occur during conidiation.

Cell Differentiation↗

PubMed1985

Isolation and characterization of genes differentially expressed during conidiation of Neurospora crassa.

A Neurospora crassa genomic DNA library was screened with a cDNA probe enriched in sequences expressed in conidiating cultures. Clones were isolated that preferentially hybridized to this probe versus a second cDNA probe complementary to polyadenylated RNA isolated from mycelia. Twelve clones contained unique sequences that hybridized to 22 transcripts, 19 of which accumulated preferentially in conidiating cultures. Eight transcripts were present in higher levels in conidiating cultures than in mycelia. Eleven transcripts were detected only in conidiating cultures and first appeared at different times during the asexual cycle. We mapped genomic sequences homologous to the 11 clones by conventional crosses using restriction fragment-length polymorphisms as genetic markers. The sequences homologous to genes expressed preferentially in conidiating cultures are distributed on six of the seven chromosomes. Clones that map to the same chromosome are linked. No recombination occurred between genomic sequences homologous to three clones, suggesting that the genes contained in these clones may constitute a gene cluster.

Cell Differentiation↗

PubMed1984

Evidence for the transcript secondary structures predicted to regulate transcription attenuation in the trp operon.

We have examined the leader transcript of the Serratia marcescens trp operon to determine if the alternate secondary structures postulated to control transcription attenuation actually form in purified trp leader RNA. RNA secondary structures were analyzed by partial digestion of 32P end-labeled leader transcripts with single and double strand-specific nucleases. We found that the 176 nucleotide wild-type transcript formed the predicted hairpin structures designated 1:2 and 3:4; the latter structure is believed to signal transcription termination. We constructed a deletion plasmid, pSm delta 1,4, to determine whether the postulated RNA antiterminator structure 2:3 could also form. This plasmid lacked the DNA regions corresponding to RNA segments 1 and 4. We found that the leader transcript from pSm delta 1,4 formed structure 2:3. This provides the first direct evidence establishing the formation of a predicted antitermination secondary structure.

Base Sequence↗

PubMed1984

Stability of an RNA secondary structure affects in vitro transcription pausing in the trp operon leader region.

Transcription of the tryptophan (trp) operon of Escherichia coli and other bacterial species is regulated by the formation of alternative secondary structures in the leader segment of the transcript. During in vitro transcription of the trp leader region RNA polymerase pauses at base pair 92 after synthesis of an RNA hairpin secondary structure. We studied the dependence of pausing on hairpin stability by examining mutant trp templates containing base pair substitutions in the region corresponding to the hairpin secondary structure. Base changes that lower the stability of the hairpin were found to reduce both the frequency and half-life of RNA polymerase pausing while base changes that do not affect hairpin stability had little effect on pausing. Pausing was enhanced by the nusA protein; this enhancement was greatly reduced on mutant templates specifying less stable hairpins. The frequency of pausing on some mutant templates was correlated with the extent of read-through transcription beyond the trp attenuator, suggesting a possible role for pausing in the coupling of transcription and translation during transcription of the leader region of the operon.

DNA-Directed RNA Polymerases↗

PubMed1984

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.

Amino Acid Sequence↗

PubMed1984

Genetic analysis of the tryptophan operon regulatory region using site-directed mutagenesis.

The regulatory region of the tryptophan operon of Escherichia coli was subjected to in vitro site-directed mutagenesis using sodium bisulfite as the mutagen. The mutagenized DNA was used to transform cells to drug resistance, and plasmid DNAs from individual transformants were isolated and sequenced. Overall, 22% of the plasmids sequenced contained alterations within the region of interest. Many of the mutants obtained had characteristics that bear on aspects of the alternative secondary structure model of attenuation. Expression analyses with several of the mutants provided evidence suggesting that ribosome dissociation at the leader peptide stop codon may be rapid and that this dissociation is responsible for setting the steady-state level of expression observed in cultures growing in the presence of excess tryptophan. One mutation altered the amino acid composition of the leader peptide without affecting a transcript secondary structure. The behavior of this mutant supports the prediction that the leader peptide per se plays no role in attenuation, rather it is the act of synthesis of the peptide that has regulatory significance. Several of the mutations provide information on the structure of the RNA antiterminator . Additional mutations support the conclusion that the last stem and loop structure in the terminated transcript, structure 3:4, is sufficient to cause transcription termination.

Anthranilate Synthase↗

PubMed1984

Use of complementary DNA oligomers to probe trp leader transcript secondary structures involved in transcription pausing and termination.

DNA oligomers were synthesized that are perfectly complementary to different segments of the tryptophan (trp) operon leader transcript. These 15 nucleotide long oligomers were used as probes of the involvement of transcript secondary structures in two processes: transcription pausing at the pause site located near base pair 90 in the leader region, and transcription termination at the attenuator. The 15-mers were complementary to the four segments of the trp leader transcript which have been shown to form the alternative secondary structures that are believed to be responsible for pausing, termination, and antitermination. Oligomers complementary to RNA segments 1 and 3 relieved termination while the 15-mer complementary to RNA segment 1 relieved pausing. 15-mers complementary to segment 2 had no effect on pausing and the oligomer complementary to segment 4 had virtually no effect on termination.

Base Sequence↗

PubMed1984

Nucleotide sequence of Saccharomyces cerevisiae genes TRP2 and TRP3 encoding bifunctional anthranilate synthase: indole-3-glycerol phosphate synthase.

Saccharomyces cerevisiae anthranilate synthase:indole-3-glycerol phosphate synthase is a multifunctional hetero-oligomeric enzyme encoded by genes TRP2 and TRP3. TRP2, encoding anthranilate synthase Component I, was cloned by complementation of a yeast trp2 mutant. The nucleotide sequence of TRP2 as well as that of TRP3 were determined. The deduced anthranilate synthase Component I primary structure from yeast exhibits only limited similarity to that of the corresponding Escherichia coli subunit encoded by trpE. On the other hand, yeast anthranilate synthase Component II and indole-3-glycerol phosphate synthase amino acid sequences from TRP3 are clearly homologous with the corresponding sequences of the E. coli trpG and trpC polypeptide segments and thereby establish the bifunctional structure of TRP3 protein. Based on comparisons of TRP3 amino acid sequence with homologous sequences from E. coli and Neurospora crassa, an 11-amino acid residue connecting segment was identified which fuses the trpG and trpC functions of the bifunctional TRP3 protein chain. These comparisons support the conclusion that the amino acid sequence of connectors in homologous multifunctional enzymes need not be conserved. Connector function is thus not dependent on a specific sequence. Nuclease S1 mapping was used to identify mRNA 5' termini. Heterogeneous 5' termini were found for both TRP2 and TRP3 mRNA. TRP2 and TRP3 5'-flanking regions were analyzed for sequences that might function in regulation of these genes by the S. cerevisiae general amino acid control system. The 9 base pair direct repeat (Hinnebusch, A.G., and Fink, G.R. (1983) J. Biol. Chem. 258, 5238-5247) and inverted repeats were identified in the 5'-flanking sequences of TRP2 and TRP3.

Amino Acid Sequence↗

PubMed1984

Comparison of regulatory and structural regions of genes of tryptophan metabolism.

The genes of tryptophan biosynthesis are arranged and regulated differently in many microorganisms. Comparison of the transcription regulatory regions of the trp operons of several species of enterobacteria reveals that those sequences and structures believed to be essential for repression and attenuation control are conserved. Examples of divergent and convergent evolutionary change are presented. Rearrangements involving the homologous trpG and pabA genes and their presumed ancestral bi-specific gene are described. Alignment of homologous sequences of trp polypeptides encoded by fused and nonfused genes from various species reveals short connecting amino acid sequences at fusion junctions. These connecting sequences may be relics of gene fusion events and/or they may facilitate the proper folding of neighboring polypeptide domains.

Amino Acid Sequence↗

PubMed1984

Repression is relieved before attenuation in the trp operon of Escherichia coli as tryptophan starvation becomes increasingly severe.

Expression of the tryptophan operon of Escherichia coli is regulated over about a 500- to 600-fold range by the combined action of repression and attenuation. Repression regulates transcription initiation in response to variation in the intracellular concentration of tryptophan. Attenuation regulates transcription termination at a site in the leader region of the operon in response to changes in the extent of charging of tRNATrp. We measured repression independently of attenuation to ascertain whether these regulatory mechanisms were used differentially by the bacterium as the severity of tryptophan starvation was increased. We found that repression regulated transcription of the operon over the range from growth with excess tryptophan to growth under moderate tryptophan starvation. By contrast, attenuation (termination control) was not relaxed until tryptophan starvation was in the moderate-to-severe range. Thus, attenuation and repression were used to regulate transcription in response to different degrees of tryptophan deprivation. Consistent with this conclusion is the observation that when tryptophan starvation was sufficient to relieve repression 50 to 60%, 65% of the tRNATrp of the bacterium was charged. These findings provide a possible explanation for the existence of only two tryptophan codons in the coding region for the trp leader peptide of Enterobacteriaceae.

Bacteriophage lambda↗

PubMed1984

Correction of the nucleotide sequence of the Citrobacter freundii tryptophan operon regulatory region.

We present a correction of the previously reported nucleotide sequence of the Citrobacter freundii trp operon regulatory region. The original sequence analyses were performed with a plasmid designated pCF2. We repeated the cloning of the trp regulatory region of C. freundii and concluded from the determined sequence that a DNA rearrangement had occurred within the leader region of the cloned trp DNA of pCF2. The correct sequence is homologous to the Escherichia coli sequence.

Amino Acid Sequence↗

PubMed1984

Overproduction of tryptophanyl-tRNA synthetase relieves transcription termination at the Escherichia coli tryptophan operon attenuator.

Overproduction of tryptophanyl-tRNA synthetase increased trp operon expression by reducing transcription termination at the trp attenuator. The total cellular level of charged tRNATrp was not affected by increased levels of the synthetase. We propose that excess synthetase binds charged tRNATrp and reduces the concentration available for translation.

Amino Acyl-tRNA Synthetases↗

PubMed1983

Functional inferences from crystals of Escherichia coli trp repressor.

We have reproducibly grown crystals of L-tryptophan . trp aporepressor and indole-3-propionate . trp aporepressor complexes from Escherichia coli which are suitable for x-ray diffraction analysis. The active repressor, L-tryptophan . aporepressor, crystallizes in both trigonal (P3(1)21 or P3(2)21) and tetragonal (P4(1)22 or P4(3)22) forms which diffract to at least 2.0 and 2.5 A, respectively. The trigonal form has one-half of the functional dimer/asymmetric unit; therefore, the trp repressor molecule has an axis of 2-fold rotational symmetry corresponding to the lattice dyad. The inactive complex, indole-3-propionate . aporepressor, or "pseudorepressor," forms tetragonal crystals that also diffract to at least 2.5 A and are isomorphous to those of the active repressor. Slight differences between their diffraction patterns indicate modest structural differences between active and inactive complexes that are presumably mediated by the alpha-amino group of L-tryptophan and account for operator-specific binding.

Apoproteins↗

PubMed1983

A complementary DNA oligomer releases a transcription pause complex.

The formation of alternative secondary structures in the transcript of the tryptophan (trp) operon leader region regulates expression of the trp operons of Escherichia coli and other bacterial species. During in vitro transcription RNA polymerase pauses near base pair 90 after the first hairpin secondary structure in E. coli trp leader mRNA is formed. The E. coli L-factor enhances transcription pausing at this site (Farnham, P. J., Greenblatt, J., and Platt, T. (1982) Cell 29, 945-951); presumably it does so by facilitating recognition of the RNA hairpin by polymerase. We show that addition of a DNA oligomer complementary to the proximal segment of the RNA hairpin relieves transcription pausing in vitro both in the presence and absence of L-factor. The oligomer apparently interferes with formation of the RNA hairpin which we believe is recognized by polymerase as the pause signal. The oligomer also relieves pausing in L-factor-induced paused complexes, suggesting that the oligomer can disrupt a preformed secondary structure in the transcript.

DNA↗

PubMed1983

Mutations of the beta subunit of RNA polymerase alter both transcription pausing and transcription termination in the trp operon leader region in vitro.

RNA polymerase was purified from rifampicin-resistant mutants of Escherichia coli which exhibit altered transcription termination at the trp operon attenuator in vivo. These mutant polymerases were used to investigate transcription pausing at the trp leader pause site and transcription termination at the trp attenuator. The mutant polymerases examined in vitro mimic their in vivo termination responses; i.e. RNA polymerase isolated from a mutant which displays high transcriptional read-through of the trp operon in vivo allows greater transcriptional read-through in vitro, while RNA polymerase prepared from a mutant which has reduced read-through in vivo exhibits greater termination of transcription in vitro. The observed differences are not due to the presence of--or response to--alternate secondary structures in the trp leader transcript since deletion of the DNA segment corresponding to some of these alternate structures does not affect termination efficiency. The mutant polymerases also have comparable effects on the kinetics of transcription pausing at the trp leader pause site; the termination-deficient polymerase exhibits diminished pausing while the termination-proficient polymerase displays enhanced pausing. We suggest that this correlation reflects polymerase recognition of similar features of RNA secondary structures in the pause and termination events. In addition, since single mutational changes in RNA polymerase affect two activities, pausing and termination, it is likely that a single site or region of the polymerase is involved in both events.

Base Sequence↗