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Biomedical subjects

C Yanofsky

Publications and source records attributed to C Yanofsky.

At least 199 records · Page 11Linked to original sources
PubMed1978

Purification and properties of a third form of anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase from the Enterobacteriaceae.

Anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase was purified from the bacterium Erwinia carotovora, a member of the Enterobacteriaceae. The enzyme was homogeneous according to the criteria of gel electrophoresis and NH2-terminal amino acid sequence analysis. The molecular weight of the enzyme as determined on a calibrated Sephadex G-200 column was 67,000 +/- 2,000. Sodium dodecyl sulfate-polyacrylamide gels gave a subunit molecular weight of 40,000 +/- 1,000, suggesting that the enzyme was a dimer. A comparison of the NH2-terminal sequence of the enzyme with the (previously determined) homologue from Serratia marcescens, a monomer with a molecular weight of 45,000, showed that the larger Serratia subunit came into register with amino acid 14 of the Erwinia subunit. The register for the length of the known overlap, 26 amino acids, was highly conserved.

Amino Acid Sequence↗
PubMed1978

Single base-pair alterations in the Escherichia coli trp operon leader region that relieve transcription termination at the trp attenuator.

We have isolated a set of regulatory mutants defective in transcription termination at the attenuator in the leader region of the Escherichia coli tryptophan (trp) operon. In vivo the mutants have 2- to 4-fold increased levels of expression of the trp operon above the level of the trpR parental strain. These levels are increased an additional 1.5- to 2-fold when the mutants are starved of tryptophan. Transcription termination at the trp attenuator was analyzed in vitro with DNA restriction fragments containing the termination-relief mutations. Whereas the frequency of readthrough transcription beyond the termination site is 5% with the wild-type DNA template, it is 46-76% when mutant DNAs are used as templates. The base change in the leader region of each mutant was determined by RNA and/or DNA sequencing. All the changes were between base pairs +116 and +132, in the G-C-rich segment of the leader region. The RNA residues between +114 and +134 of the leader transcript can form a stable stem and loop structure [deltaG approximately equal to -20 kcal (-84 kJ)]. All of the termination-relief mutations destabilize this structure (deltaG approximately equal to -9.0 to -10.5 kcal). These results suggest that the efficiency of transcription termination may be dependent on the integrity of the secondary structure of the above segment of the transcript of the leader region.

Base Sequence↗
PubMed1978

Naturally occurring promoter down mutation: nucleotide sequence of the trp promoter/operator/leader region of Shigella dysenteriae 16.

The promoter/operator/leader region of the trp operon of Shigella dysenteriae 16 has single base pair differences from the corresponding region of Escherichia coli at positions -24 and -13. The difference at -13 was shown to be responsible for the 90% reduction in promoter function characteristic of the trp operon of S. dysenteriae. The base pair difference at position -13 also renders the operator partially constitutive. This allows the organism to maintain relatively high repressed levels of the trp enzymes and increases the relative importance of attenuation as a transcription control mechanism. These findings and the earlier observation that the trpE protein of S. dysenteriae is only slightly active explain the low in vivo expression of the trp operon of this organism. Nutritional studies suggest that operons involved in other amino acid biosynthetic pathways in S. dysenteriae 16 may be similarly partially inactivated.

Base Sequence↗
PubMed1978

Translational control of transcription termination at the attenuator of the Escherichia coli tryptophan operon.

We have isolated two regulatory mutants altered in the leader region of the Escherichia coli tryptophan (trp) operon. In one mutant, trpL29, the AUG translation start codon for the trip leader peptide is replaced by AUA. The other mutant, trpL75, has a G leads to A change at residue 75, immediately after the UGA translation stop codon for the trp leader peptide. In vivo, trpL29 and trpL75 increase the efficiency of transcription termination at the trp attenuator 3- to 5-fold. trpL29 and trpL75 also fail to respond fully to tryptophan starvation and other conditions that normally relieve transcription termination at the trp attenuator. The trpL29 mutation, which presumably reduces synthesis of the trp leader peptide, is cis dominant. The effect of starvation for a number of the amino acids in the trp leader peptide was determined. Only starvation for tryptophan and arginine, amino acids that occur at residues 10, 11, and 12 of the 14-residue trp leader peptide, elicits relief of transcription termination. Our findings suggest that translation of trp leader RNA is involved in regulation of transcription termination at the attenuator. A model is discussed in which the location of the ribosome synthesizing the leader peptide is communicated to the RNA polymerase transcribing the leader region.

Base Sequence↗
PubMed1978

Nucleotide sequence of the leader region of the phenylalanine operon of Escherichia coli.

The pheA structural gene of the phenylalanine operon of Escherichia coli is preceded by a transcribed leader region of about 170 nucleotide pairs. In vitro transcription of plasmids and restriction fragments containing the phe promoter and leader region yields a major RNA transcript about 140 nucleotides in length. This transcript, pheA leader RNA, has the following features: (i) a potential ribosome binding site and AUG translation start codon about 20 nucleotides from its 5' end; (ii) 14 additional in phase amino acid codons and a UGA stop codon after the AUG; 7 of these 14 are Phe codons; (iii) a 3'-OH terminus about 140 nucleotides from the 5' end (transcription termination occurs in an A.T-rich region which is subsequent to a G.C-rich region; just beyond the site of transcription termination there is a sequence corresponding to a ribosome binding site and the AUG translation start codon of the pheA structural gene); (iv) a sequence which would permit extensive intrastrand stable hydrogen bonding. In addition to G.C-rich stem structures, highly analogous to those proposed for the leader RNAs of the tryptophan operons of E. coli and Salmonella typhimurium [Lee, F. & Yanofsky, C. (1977) Proc. Natl. Acad. Sci. USA 74, 4365-4369], there is also extensive base-pairing possible between the phe codon region and a more distal region of the leader transcript. The roles of synthesis of the Phe-rich leader peptide and secondary structure of the leader transcript in the regulation of transcription termination at the attenuator of the phe operon are discussed.

Base Sequence↗
PubMed1978

Translation of the leader region of the Escherichia coli tryptophan operon.

When the trp operon of Escherichia coli contains either of two deletions that fuse the initial portion of the leader region to the distal segments of the trpE gene, novel fusion polypeptides are produced. The new polypeptides are synthesized efficiently both in vivo and in vitro, and their synthesis is subject to repression by trp repressor. Fingerprint analyses of tryptic and chymotryptic digests of the new polypeptides show that both contain trpE polypeptide sequences and, despite their different sizes, share the same N-terminal sequence. Our results suggest that synthesis of the new polypeptides is initiated at the AUG-centered ribosome-binding site in the leader region and proceeds in phase to the region coding for the C-terminal end of the trpE polypeptide.

Bacterial Proteins↗
PubMed1977

Structure and properties of a hybrid tryptophan synthetase of alpha chain produced by genetic exchange between Escherichia coli and Salmonella typhimurium.

Genetic exchange between the structural genes for the alpha chain of tryptophan synthetase [tryptophan synthase; L-serine hydro-lyase (adding indoleglycerol-phosphate), EC 4.2.1.20] of E. coli and S. typhimurium yielded recombinant genes that specified functional hybrid polypeptides. The alpha chains produced by three recombinants appeared to be identical but differed from those of E. coli and S. typhimurium by at least 27 and 8 amino acid residues, respectively. In vivo and in vitro tests of enzyme function suggest that the hybrid alpha chains are near-equivalent to their fully active parental proteins.

Amino Acids↗
PubMed1977

Transcription termination at the trp operon attenuators of Escherichia coli and Salmonella typhimurium: RNA secondary structure and regulation of termination.

Transcription termination at the attenuators of the trp operons of Escherichia coli and Salmonella typhimurium was studied in vitro using DNA restriction fragments as templates. Readthrough transcription beyond the terminators occurred with 5 and 30% efficiency, respectively, in E. coli and S. typhimurium. This difference is correlated with the stability of proposed secondary structures of the respective trp leader transcripts. Secondary structure analyses of the two leader transcripts revealed a well-conserved pattern of RNA base paring. This and the possibility that trp leader RNA is translated suggest a model for regulation of transcription termination that is based on ribosome movement along the RNA and a shift between alternative RNA base-pairing configuration.

Escherichia coli↗
PubMed1977

Mitomycin C-induced expression of trpA of Salmonella typhimurium inserted into the plasmid ColE1.

EcoRI endonuclease digestion of the deoxyribonucleic acid of a phi80 transducing phage carrying the entire tryptophan (trp) operon of Salmonella typhimurium (phi80 S.t.trpE-A) yielded a 4.3 X 10(6)-dalton fragment containing intact trpE, trpD, and trpC and a 3.35 X 10(6)-dalton fragment containing intact trpA. The trpA fragment inserted into EcoRI-cleaved plasmids ColE1 and CR1 was expressed regardless of its orientation of insertion. Mitomycin C, a compound that induces colicin E1 production in ColE1-containing bacteria, stimulated tryptophan synthetase alpha production in cells containing ColE1-TRPA plasmids with the trpA fragment inserted in one orientation but not the other. We conclude that in the inducible plasmids trpA can be expressed from the colicin E1 promoter.

Colicins↗
PubMed1976

Purification, subunit structure and partial amino-acid sequence of anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase from the enteric bacterium Serratia marcescens.

The enzyme anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase from Serratia marcescens was purified to apparent homogeneity. The purification procedure included ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex gel filtration and hydroxyapatite chromatography. The molecular weight of the native protein as determined on a calibrated Sephadex G-200 column was 45000. Dodecylsulfate-polyacrylamide gel electrophoresis in the presence of reducing agent revealed a subunit molecular weight of 43000 +/- 900, suggesting that the enzyme exists as a monomer. The sequence of the amino-terminal 38 residues revealed that three amino amino acids, glutamine (six residues), glutamic acid (five residues) and serine (five residues) comprised 42% of the sequence composition.

Amino Acid Sequence↗