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G Zurawski

Publications and source records attributed to G Zurawski.

64 records · Page 4Linked to original sources
PubMed1979

The nucleotide sequence preceding and including the beginning of the ilvE gene of the ilvGEDA operon of Escherichia coli K12.

The DNA sequence of the 570 base pairs that precedes and includes the beginning of the ilvE gene shows no evidence of a leader-attenuator region. Instead, the sequence shows that the ilvE gene is preceded by another structural gene, presumably the normally cryptic ilvG gene. In vitro transcription of the two plasmids (pLC26-3 and pRL5) containing the regulatory region of the ilvEDA operon results in the formation of two "leader" RNAs. These results are consistent with the suggestion that the ilvEDA operon is regulated by an attenuator mechanism and that the attenuator region lies before the ilvG gene.

Base Composition↗
PubMed1979

Attenuation in the Escherichia coli tryptophan operon: role of RNA secondary structure involving the tryptophan codon region.

The secondary structure of the terminated trp leader transcript from Escherichia coli was analyzed by RNase T1 partial digestion. Base-paired regions were recovered by nondenaturing gel electrophoresis and identified by denaturing gel electrophoresis and fingerprinting. The tandem tryptophan codons in the leader peptide coding region were found to be base paired with a more distal region of the transcript. This and other secondary structures that the trp leader RNA can form help explain the physiological response of the operon as well as the behavior of regulatory mutants.

Codon↗
PubMed1979

Structural and functional analysis of cloned deoxyribonucleic acid containing the trpR-thr region of the Escherichia coli chromosome.

Specialized transducing phages containing the thr-trpR region of the Escherichia coli chromosome were derived from a strain with lambda prophage inserted in thr. Cloning of segments of the chromosomal deoxyribonucleic acid of one such lambda thr + trpR+ phage in various plasmid vectors established that a 1.3-kilobase BamHI fragment carried trpR+ intact. Strains with a multicopy plasmid vector containing the BamHI insert produced 20-fold-higher levels of trp aporepressor than did the wild-type strain of Escherichia coli. Similarly, induction of lambda thr + trpR+ lysogens resulted in increased aporepressor levels. The 1.3-kilobase trpR+ BamHI fragment was inserted in either orientation downstream from lambda pLN in a plasmid vector in which transcription from lambda pL was under the control of a temperature-sensitive lambda repressor. Induction established the orientation of transcription of trpR and led to the production of 100-fold-increased levels of trp aporepressor. A presumptive 23,500-dalton trpR+ polypeptide was detected by using lambda pLNtrpR+ plasmid deoxyribonucleic acid in a cell-free transcription-translation system.

Bacterial Proteins↗
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

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↗
PubMed1975

Chorismate mutase of Chlamydomonas reinhardi. Partial purification and some properties.

Chorismate mutase (chorismate pyruvatemutase, EC 5.4.99.5) was extracted from Chlamydomonas reinhardi by sonication. Fractionation of crude sonic extracts with (NH4)2SO4 and by DEAE-cellulose and Sephadex gel chromatography indicated a single peak of chorismate mutase activity with molecular weight 61 000. The Michaelis constant for 20-fold purified enzyme was 0.46 mM. Prephenate dehydrogenase (EC 1.3.1.9) and prephenate dehydratase (EC 4.2.1.40) activities were not detected in our crude or partially purified preparations of chorismate mutase. Tyrosine (1.25 mM) inhibited chorismate mutase activity by approx. 85% in crude and partially purified preparations. Phenylalanine (1.25 mM) inhibited 20%. Tryptophan (1.25 mM) by itself had no detectable effect on chorismate mutase activity but it completely reversed inhibition by tyrosine and phenylalanine. No repression of chorismate mutase was observed when the minimal growth medium was supplemented with aromatic end products.

Chlamydomonas↗