Prokaryotic mechanisms in eukaryotes?
Explore the source record for details and available documents.
Escherichia coli↗
Biomedical subjects
C Yanofsky
Publications and source records attributed to C Yanofsky.
Release of transcript and template during transcription termination at the trp operon attenuator.
We studied release of trp leader RNA and trp template DNA from RNA polymerase during transcription termination at the attenuator of the trp operon of Escherichia coli. Preliminary evidence had suggested that a stable ternary complex was formed at the trp attentuator. We observed that the complexes between RNA polymerase and trp leader RNA and the DNA template produced during transcription were labile at high salt concentrations and were undetectable when transcription was performed in the presence of heparin. These characteristics are atypical of the stable transcription termination complexes described by others (Richardson, J. P., and Conaway, R. (1980) Biochemistry 19, 4293-4299; Shigesada, K., and Wu, C. (1980) Nucleic Acids Res. 8, 3355-3369). We successfully reconstituted polymerase-trp leader RNA complexes in simple mixing experiments; these and other studies indicated that it is core polymerase that binds the leader transcript and the DNA template. In agreement with this conclusion, it was observed that sigma factor inhibited binding of RNA polymerase to the trp leader transcript and the DNA template and displaced leader RNA from RNA polymerase during transcription. It seems likely that small amounts of core polymerase present in the holoenzyme preparation, or generated during transcription, are responsible for the nonspecific binding of RNA transcript and DNA template. Our findings, therefore, suggest that the transcription termination event at the trp attenuator normally involves spontaneous dissociation of polymerase, template, and RNA transcript.
DNA, Bacterial↗
Plasmids containing the trp promoters of Escherichia coli and Serratia marcescens and their use in expressing cloned genes.
Explore the source record for details and available documents.
Amino Acid Sequence↗
In vitro synthesis of the tryptophan operon leader peptides of Escherichia coli, Serratia marcescens, and Salmonella typhimurium.
We used an in vitro DNA-dependent protein-synthesizing system to demonstrate de novo synthesis of the leader peptide specified by the tryptophan (trp) operons of several bacterial species. Peptide synthesis was directed by self-ligated short restriction fragments containing the trp promoter and leader regions. Synthesis of leader peptides was established by demonstrating that they were labeled in vitro only by those amino acids predicted to be present in the peptides. Leader peptide synthesis was abolished by the addition of the Escherichia coli trp repressor. The E. coli trp leader peptide was found to be extremely labile in vitro; it had a half-life of 3-4 min. In a highly purified DNA-dependent peptide-synthesizing system, synthesis of the di- and tripeptides predicted from the Salmonella typhimurium trp operon leader sequence, fMet-Ala and fMet-Ala-Ala, also was observed. Using this dipeptide synthesis system, we demonstrated that translation initiation at the ribosome binding site used for trp leader peptide synthesis was reduced 10-fold when the transcript contained a segment complementary to the ribosome binding site.
Bacterial Proteins↗
Purification and characterization of trp aporepressor.
We have isolated homogeneous trp aporepressor from an overproducing strain of Escherichia coli carrying a plasmid containing trpR preceded by tandem trp operon promoters. Dye-affinity and ion-exchange chromatography were used in conjunction with a gel electrophoresis assay in which the repressor, when bound to the trp operator, protects an Rsa I restriction site from endonuclease cleavage. Crystals suitable for x-ray diffraction studies were grown from a variety of concentrated salt solutions. Hydrodynamic properties and electrophoretic analysis of unmodified and covalently crosslinked aporepressor show that the free aporepressor has an isoelectric point of 5.9 and is a dimer containing two identical 12.5-kilodalton subunits in the presence or absence of L-tryptophan. The repressor . operator complex binds poorly to nitrocellulose filters, but restriction-site protection studies indicate that, in the presence of tryptophan, one dimer is bound to the operator site with an apparent dissociation constant less than 2 X 10(-9) M. Preliminary equilibrium dialysis experiments suggest that tryptophan binds to the aporepressor with a dissociation constant of 1.6 X 10(-5) M.
Amino Acid Sequence↗
Transcription termination in vitro at the tryptophan operon attenuator is controlled by secondary structures in the leader transcript.
The role of alternative RNA secondary structures in regulating transcription termination at the attenuator of the tryptophan (trp) operon of Serratia marcescens was examined in vitro by transcribing mutant DNA templates having deletions of different segments of the trp leader region. Deletions that removed sequences corresponding to successive segments of postulated RNA secondary structures either increased or decreased transcription termination at the attenuator. The results obtained are consistent with the hypothesis that transcription termination results from RNA polymerase recognition of a particular RNA secondary structure, the terminator. This structure forms only in the absence of an alternative, preceding, RNA secondary structure, the antiterminator.
Chromosome Deletion↗
Structure of the trifunctional trp-1 gene from Neurospora crassa and its aberrant expression in Escherichia coli.
The trifunctional trp-1 gene from Neurospora crassa was cloned by complementation of a phosphoribosylanthranilate isomerase-deficient mutant of E. coli. A 2.7-kb DNA sequence containing trp-1 was determined. Homology of the deduced trp-1 polypeptide sequence to the corresponding E. coli proteins is striking; the order of functional domains within trp-1 is NH2-glutamine amidotransferase-indoleglycerolphosphate synthase-phosphoribosylanthranilate isomerase-COOH (NH2-trpG-trpC-trpF-COOH). Whereas trpF complementing activity can be detected in E. coli, trpC activity is absent. It is likely that translation of trp-1 does not proceed from the proper start site in E. coli; the carboxy terminal portion of the trp-1 polypeptide may be the only portion synthesized. Fusion of a bacterial amino terminus and ribosome binding site to the trp-1 coding region results in expression of trpC as well as trpF activity in E. coli. The locations of several startpoints for trp-1 mRNA synthesis were determined by the S1 nuclease mapping technique. DNA immediately 5' to the trp-1 transcription initiation region does not possess a sequence resembling the canonical TATAAA of eukaryotes.
Base Sequence↗
Regulation of tryptophan operon expression by attenuation in cell-free extracts of Escherichia coli.
Expression of the tryptophan (trp) operon of Escherichia coli was shown to be regulated by attenuation in an in vitro DNA-dependent protein-synthesizing system. In extracts prepared from a temperature-sensitive tryptophanyl-tRNA synthetase mutant, plasmid-directed trpE enzyme synthesis was inhibited 2- to 3-fold by addition of purified wild type tryptophanyl-tRNA synthetase. When the extract used from a strain bearing a trpTts mutation that reduces charging of tRNATrp in vivo, a 2- to 3-fold increase in trpE enzyme synthesis was observed when an excess of uncharged wild type tRNATrp was added. Analysis of attenuation by measurement of trp mRNA synthesis was facilitated by constructing a plasmid (pAD1) containing the rpoC transcription terminator inserted early in trpE. Transcription proceeding past the trp attenuator of this plasmid terminates at this new terminator sequence and results in the production of a approximately 400-nucleotide long read-through transcript. Using this plasmid and extracts prepared from the tryptophanyl-tRNA synthetase mutant, a 4- to 8-fold decrease in relative read-through transcription was observed in response to exogenously added wild type tryptophanyl-tRNA synthase. Kinetic analyses of trp mRNA synthesis and studies using plasmid template DNAs bearing trp attenuator mutations indicate that translation of the leader peptide coding region of the transcript regulates transcription termination at the trp attenuator.
Cell-Free System↗
Transcript secondary structures regulate transcription termination at the attenuator of S. marcescens tryptophan operon.
We have analysed the regulatory behaviour of deletion mutants lacking different segments of the leader region of the tryptophan operon of Serratia marcescens. Our results support the model in which a particular RNA structure, the terminator, is recognized during transcription as a transcription termination signal, and an alternative RNA structure, the anti-terminator, prevents formation of the terminator. It appears that the role of translation, ribosome stalling and shifts between alternative RNA secondary structures, is simply to regulate formation of the terminator.
Base Sequence↗
Superattenuation in the tryptophan operon of Serratia marcescens.
Deletions were generated in vitro in the leader region of the tryptophan operon of Serratia marcescens and subsequently incorporated into the Escherichia coli chromosome in single copy form. Deletions which removed the translation start codon for the leader peptide or which ended in the ribosome recognition region preceding the leader peptide coding segment, caused superattenuation, that is, increased transcription termination at the attenuator. Apparently, the capacity to initiate translation of this coding region modulates expression of the operon.
Base Sequence↗
The nucleotide sequence of the structural gene for Escherichia coli tryptophanyl-tRNA synthetase.
The complete nucleotide sequence of trpS, the structural gene for Escherichia coli tryptophanyl-tRNA synthetase, was determined using a plasmid carrying the structural gene. From the single open reading frame of correct length and orientation we deduced an amino acid sequence consistent with the amino acid composition of the purified protein. In addition, previously sequenced peptides representing 52% of the protein were readily aligned with regions of the deduced sequence. The deduced amino acid sequence of the E. coli enzyme is 60% homologous with the sequence of the enzyme from Bacillus stearothermophilus. Using currently available procedures we predicted the secondary structure for the enzyme from each organism and compared these structures to those of the two aminoacyl-tRNA synthetases whose three-dimensional structures have been determined. We used a convenient plasmid recombination procedure to map the regional locations of missense mutations within trpS that have characteristic effects on the properties of the enzyme.
Amino Acid Sequence↗
Nucleotide sequence of Escherichia coli purF and deduced amino acid sequence of glutamine phosphoribosylpyrophosphate amidotransferase.
The Escherichia coli gene purF, coding for 5-phosphoribosylamine:glutamine pyrophosphate phosphoribosyltransferase (amidophosphoribosyltransferase) was subcloned from a ColE1-purF plasmid into pBR322. Amidophosphoribosyltransferase levels were elevated more than 5-fold in the ColE1-purF plasmid-bearing strain compared to the wild type control, and a further 10- to 13-fold elevation was observed in several pBR322 derivatives. The nucleotide sequence of a 2478-base pair PvuI-HinfI fragment encoding purF was determined. The purF45 structural gene codes for a 56,395 Mr protein chain having 504 amino acid residues. Methionine-1 is removed by processing in vivo leaving cysteine as the NH2-terminal residue. The deduced amino acid sequence was confirmed by comparisons with the NH2-terminal amino acid sequence determined by automated Edman degradation (Tso, J. Y., Hermodson, M. A., and Zalkin, H. (1982) J. Biol. Chem. 257, 3532-3536) and amino acid analyses of CNBr peptides including a 4-residue peptide from the CO2H terminus of the enzyme. Nucleotide sequences characteristic of bacterial promoter-operator regions were identified in the 5' flanking region. The coding region appears to be preceded by a 277-297 nucleotide mRNA leader. A deletion removing the putative promoter-operator region results in defective purF expression.
Amidophosphoribosyltransferase↗
Nucleotide sequence of trpE of Salmonella typhimurium and its homology with the corresponding sequence of Escherichia coli.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Yeast gene TRP5: structure, function, regulation.
The nucleotide sequence of the yeast gene TRP5 and its 5' and 3' flanking regions was determined. The deduced coding sequence for tryptophan synthase contains 2,127 base pairs. The protein chain has a calculated molecular weight of 76,544. Yeast tryptophan synthase, a bifunctional protein, has a primary structure which corresponds to an Escherichia coli tryptophan synthase alpha chain-beta chain fusion. An NH2-terminal 239 amino acid segment of yeast tryptophan synthase is homologous with E. coli tryptophan synthase alpha subunit, while a distal 389 amino acid residue segment is homologous to the E. coli tryptophan synthase beta chain. This order of segments of the yeast enzyme is the reverse of the chromosomal order characteristic of all prokaryotes that have been examined. The two segments are joined by a connecting region of 28 residues in the yeast enzyme which is not homologous to either the alpha or beta chains of the bacterial enzyme. A portion of the connecting region of yeast tryptophan synthase exhibits nucleotide sequence similarity to the 3' terminus of E. coli trpC and the trpC-trpB intercistronic region. Active site cysteine, histidine, and lysine residues in the beta 2 subunit of E. coli tryptophan synthase are conserved in the yeast enzyme. Also conserved in the yeast enzyme are 6/8 amino acid residues having an important role in maintaining the structure and function of the E. coli tryptophan synthase alpha subunit. S1 nuclease mapping was used to identify three major mRNA transcripts with different 5' termini. Potential T-A-T-A sites for transcription initiation were identified, as well as other sequences that occur frequently in yeast genes. A 5' flanking region of TRP5 was shown by DNA/DNA hybridization to be present in multiple copies in the yeast genome. TRP5 mRNA levels, measured by RNA/DNA hybridization, increased 2- to 7-fold in response to starvation for either tryptophan or histidine, indicating transcriptional regulation.
Amino Acid Sequence↗
Trp aporepressor production is controlled by autogenous regulation and inefficient translation.
We constructed a trpR-lacZ gene fusion that specifies a hybrid protein that has full beta-galactosidase activity. The gene fusion was associated with the unaltered trpR transcription and translation control region; thus, hybrid beta-galactosidase production was an indicator of expression of the trp aporepressor (trpR) operon. To facilitate in vivo expression studies, a DNA segment containing the trpR-lacZ gene fusion and the trpR controlling region was transferred to bacteriophage lambda and subsequently inserted into the bacterial chromosome. Analyses of hybrid beta-galactosidase production showed that the trpR operon is regulated autogenously but that the rate of synthesis of aporepressor varies only 4- to 5-fold in response to changes in the intracellular concentration of tryptophan. Under comparable conditions, the trp operon is regulated by trp repressor approximately 70-fold. Therefore, the operators of the trp operon and the trpR operon must have very different affinities for trp repressor in vivo. The promoter controlling trpR expression was found to be moderately active. Nevertheless, there are only about 50-300 molecules of trp aporepressor per cell. The low aporepressor level appears to be due to inefficient translation of trpR mRNA.
Apoproteins↗
Transcription termination at the tryptophan operon attenuator is decreased in vitro by an oligomer complementary to a segment of the leader transcript.
A DNA oligomer 15 nucleotides long was used to probe the involvement of RNA secondary structure in the control of transcription termination at the attenuator of the tryptophan (trp) operon of Escherichia coli. This 15-mer is perfectly complementary to a segment of trp RNA that is thought to play a role in regulation of attenuation. When added to an in vitro transcription reaction mixture containing wild-type E. coli or Salmonella typhimurium trp operon templates, the complementary 15-mer caused a 4-fold increase in read-through transcription. By contrast, the 15-mer did not affect attenuation when a mutant E. coli template was used that does not allow formation of a crucial RNA secondary structure. Control experiments established that oligomers that were not complementary to E. coli trp leader RNA did not affect attenuation and that the 15-mer did not reduce termination when the transcript lacked a complementary region. Other experiments established that the 15-mer did not increase read-through transcription by allowing RNA polymerase molecules that might have already stopped at the attenuator to resume transcription. These findings provide direct support for the view that alternate base-paired structures control transcription termination at the trp attenuator.
DNA, Bacterial↗
Regulation of tryptophanyl-tRNA synthetase formation.
A previously constructed trp-S-lacZ fusion encoding a hybrid protein with beta-galactosidase activity was subcloned from a multicopy plasmid onto a lambda vector. Single-copy lysogens of lambda trpS-lacZ were used to determine whether trpS was regulated in a manner similar to that of other aminoacyl-tRNA synthetases. trpS regulation was found to resemble that of the majority of synthetases, in that expression of the lysogen-encoded hybrid beta-galactosidase varied with growth rate; beta-galactosidase activity increased 2.5-fold as the generation time decreased from 150 to 37 min. This regulatory response was confirmed by DNA/RNA hybridization experiments, which also suggested that this form of metabolic regulation occurred at the transcriptional level. No alteration in the level of hybrid beta-galactosidase was observed, however, when cells were starved for tryptophan.
Amino Acyl-tRNA Synthetases↗
Transcription initiation at the tryptophanase promoter of Escherichia coli K-12.
Restriction fragments containing the region preceding the tryptophanase structural gene, tnaA, were used as templates for in vitro transcription experiments. A transcription initiation site was detected that was dependent on the catabolite gene activator protein (CAP) plus cyclic AMP (cAMP). The mRNA produced in vitro was fingerprinted, and the nucleotide at which transcription was initiated was localized to the vicinity of two guanine residues 316 and 318 base pairs upstream of tnaA. A region exhibiting extensive difold symmetry and homology to the CAP binding site adjacent to the lactose operon promoter exists approximately 60 base pairs preceding the site of transcription initiation. Two HinfI restriction sites are located in this region. Restriction enzyme cleavage at these sites was prevented when DNA containing the promoter region was preincubated with CAP and cAMP. RNA polymerase was incapable of protecting these sites against this cleavage. CAP and cAMP addition did not protect against cleavage at a DdeI restriction site located in the -20 region of the promoter. RNA polymerase did protect against DdeI cleavage but only in the presence of CAP and cAMP. Thus, transcription initiation at the tryptophanase promoter involves cAMP-dependent, CAP-facilitated binding of RNA polymerase to the DNA.
Base Sequence↗