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PubMed1972

Detection and isolation of the repressor protein for the tryptophan operon of Escherichia coli.

DNA from a transducing bacteriophage carrying a fusion of the tryptophan and lactose operons of E. coli (lambdadtrp-lac) has been used to direct cell-free synthesis of beta-galactosidase (EC 3.2.1.23). Whereas normal lac operon (lambdadlac) DNA requires adenosine-3':5'-cyclic monophosphate (cAMP) for beta-galactosidase synthesis, trp-lac DNA is unaffected by cAMP. This difference in cAMP dependence verifies the presence of a cAMP-requiring promoter in the lac operon that has been removed from the trp-lac DNA. Synthesis with trp-lac DNA is controlled by the protein product of the tryptophan repressor gene (trpR). Synthesis in extracts of trpR(-) (repressor-negative) cells is progressively reduced by increased additions of extract from trpR(+) cells. No trpR(-) product repression is seen when beta-galactosidase synthesis is programmed by normal lac DNA. This highly sensitive and specific assay has facilitated quantitation and partial purification of the trp repressor.

Bacterial Proteins↗

PubMed1973

Nucleotide sequences from tryptophan messenger RNA of Escherichia coli: the sequence corresponding to the amino-terminal region of the first polypeptide specified by the operon.

Mutants with internal deletions that terminate near the operator end of the tryptophan operon of E. coli were used in studies on the nucleotide sequence at the 5' end of the messenger RNA transcribed on this operon. The findings obtained permitted identification of a sequence of 43 nucleotides corresponding to the aminoterminal 11 amino acids of anthranilate synthetase component I, the polypeptide specified by the operator-proximal structural gene of the operon. It was also shown that the translation initiation codon for this polypeptide is preceded on the messenger by a "leader" sequence of unknown function, at least 150 nucleotides in length.

Amino Acid Sequence↗

PubMed1975

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp): positive effector for histidine operon transcription and general signal for amino-acid deficiency.

Maximal expression of the histidine operon of Salmonella typhimurium in a coupled in vitro transcription-translation system is strongly dependent upon addition of guanosine 5'-diphosphate 3'-diphosphate (ppGpp). This requirement for ppGpp is exerted at the level of transcription through a mechanism distinct from the his-operon-specific regulatory mechanism. In vivo derepression of the his operon is markedly defective when histidine starvation is imposed on a relA mutant--unable to rapidly increase synthesis of ppGpp--growing in amino-acid-rich medium. Increased sensitivity of relA mutants to growth inhibition by a number of amino-acid analogs suggests that ppGpp is generally important in adjusting expression of amino-acid-producing systems. Analysis of these findings leads us to propose that ppGpp is a positive effector in a system that enables the cell to balance endogenous amino-acid production with environmental conditions of amino-acid availability, and to compensate efficiently for transient changes in these conditions. We propose a unifying theory of the role of ppGpp as the general signal molecule (alarmone) in a "super-control" which senses an amino-acid deficiency and redirects the cell's economy in response.

Amino Acids↗

PubMed1975

Regulation of histidine operon does not require hisG enzyme.

Mutations are described which delete all or part of the first structural gene (hisG) of the histidine operon of Salmonella typhimurium. Physiological regulation of histidine enzymes occurs normally in strains carrying any deletion that has both endpoints within the hisG gene. Constitutive high operon expression is observed in strains carrying any hisG deletion and an unlinked regulatory mutation, hisT1504. These results strongly indicate that the hisG protein is not an essential component of the mechanism for regulating expression of the histidine operon.

Cell Division↗

PubMed1978

Transcription termination: nucleotide sequence at 3' end of tryptophan operon in Escherichia coli.

We have determined the RNA and DNA sequences in the region specifying termination of transcription at the end of the tryptophan (trp) operon of Escherichia coli. A 3'-terminal mRNA fragment of about 150 nucleotides yielded oligonucleotide products that could be assigned to the end of trpA (the last structural gene in the operon) by correlation with the amino acid sequence of the protein product. Analysis of the DNA corresponding to this region served to align the few noncoding RNA oligonucleotide sequences and demonstrated that termination of trp transcription occurs in vivo at a site 36 nucleotides after trpA, with greater than 95% efficiency. In two different strains partially defective in the transcription termination factor rho, the purified transcript is much longer and more complex, suggesting that a significant amount of read-through occurs in these strains. This is consistent with evidence [Guarente, L. P., Mitchell, D. H. & Beckwith, J. (1977) J. Mol. Biol. 112, 423-436] that efficient termination in vivo at the end of the trp operon is a rho-dependent event. The trp terminator (trp t) shares several features with other known sites of transcription termination, including (i) a 3'-terminal RNA sequence of several uridine residues, C-A-U-U-U-U(OH), (ii) a G.C-rich region in the DNA immediately preceding the site of termination, followed by an A.T-rich region, and (iii) a region of dyad symmetry in the DNA which, in the transcript, is capable of forming a stable hairpin containing seven G.C base pairs and one A.U base pair in its stem.

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

DNA sequence from the histidine operon control region: seven histidine codons in a row.

The DNA sequence of 250 base pairs preceding the first structural gene of the histidine operon of Salmonella typhimurium was determined by the dideoxy chain-termination method. Single-stranded DNA template was provided by an M13-histidine transducing phage constructed for the purpose by in vitro recombination. The termination site for the histidine leader RNA is identified by analogy with the trp operon leader termination sequence, and is 47 nucleotides before the start codon of the first structural gene G. Beginning 150 nucleotides before the end of the presumed leader RNA is a possible short protein-coding region with seven histidine codons in a row. It is proposed that the major mechanism of histodine operon control must involve a ribosome arrested at this run of histidine codons when histidine is limiting.

Base Sequence↗

PubMed1979

leu operon of Salmonella typhimurium is controlled by an attenuation mechanism.

The nucleotide sequence of the control region of the leu operon of Salmonella typhimurium was determined. A prominent feature of this region is a signal for termination of transcription. In vitro, transcription does terminate at this site, yielding a leader RNA of about 160 nucleotides as a major product. This leader RNA is potentially translatable into a peptide containing 28 amino acids, 4 of which are adjacent leucine residues. Several regions of base complementarity exist within the leader, positioned such that pairing of one region precludes pairing of another. The position of the four leucine codons relative to two regions of base complementarity suggest a model for the regulation of the leu operon similar to that proposed by Yanofsky and coworkers for the trp operon. In addition, a third region of base complementarity was identified which, when incorporated into the model, explains why premature termination is the usual outcome when transcription is initiated in vitro by purified RNA polymerase.

Base Sequence↗

PubMed1979

Regulation of the threonine operon: tandem threonine and isoleucine codons in the control region and translational control of transcription termination.

The DNA sequence of 178 base pairs preceding the first structural gene of the threonine operon of Escherichia coli has been determined. A region of perfect 2-fold rotational symmetry, involving 28 base pairs, precedes the first structural gene. The structural similarity of this sequence to known RNA polymerase termination sites suggests that this region is the termination site of the threonine operon leader RNA. Moreover a mutation (thr 79-20), which confers a depressed, constitutive phenotype, was sequenced and found to be a G.C insertion in the putative terminator. A potential coding region for a 21-amino acid leader peptide ends approximately 18 base pairs before the terminator. This peptide contains eight threonine and four isoleucine codons. Eleven of these codons are in tandem. A model for threonine operon regulation, involving alternative secondary RNA structures and translation of leader RNA, is discussed.

Base Sequence↗

PubMed1980

Model for regulation of the histidine operon of Salmonella.

A model is proposed that accounts for regulation of the histidine operon by a mechanism involving alternative configuration of mRNA secondary structure (the alternative stem model). New evidence for the model includes sequence data on three regulatory mutations. The first (hisO1242) is a mutation that deletes sequences needed to form the attenuator mRNA stem and causes constitutive operon expression. The second mutation (hisO9654) is a His- ochre (UAA) mutation in the leader peptide gene; the existence of this mutation constitutes evidence that the leader peptide gene is translated. The third mutation (hisO9663) is remarkable. It neither generates a nonsense codon nor affects a translated sequence; yet, it is suppressible by amber suppressors. We believe this mutation causes a His- phenotype by interfering with mRNA secondary structure. The suppressibility of the mutation is probably due to disruption of the attenuator stem by ribosomes that read through the terminator codon of the leader peptide gene. This explanation is supported by the observation of derepression of a wild-type control region in the presence of an amber suppressor. Evidence is presented that hisT mutants (which lack pseudouridine in the anticodon arm of histidine tRNA) may cause derepression of the his operon by slowing protein synthesis in the leader peptide gene.

Bacterial Proteins↗

PubMed1982

Transcriptional activity of the transposable element Tn10 in the Salmonella typhimurium ilvGEDA operon.

Polarity of Tn10 insertion mutations in the Salmonella typhimurium ilvGEDA operon depends on both the location and the orientation of the Tn10 element. One orientation of Tn10 insertions in ilvG and ilvE permits low-level expression of the downstream ilvEDA and ilvDA genes, respectively. Our analysis of Salmonella ilv recombinant plasmids shows that this residual ilv expression must result from Tn10-directed transcription and does not reflect the presence of internal promoters in the ilvGEDA operon, as was previously suggested. The opposite orientation of Tn10 insertion in ilvE prevents ilvDA expression, indicating that only one end of Tn10 is normally active in transcribing adjacent genes. Both orientations of Tn10 insertion in ilvD exert absolute polarity on ilvA expression. Expression of ilvA is known to be dependent on effective translation of ilvD, perhaps reflecting the lack of a ribosome binding site proximal to the ilvA sequence. Therefore, recognition of the ability of Tn10 to promote transcription of contiguous genes in the ilvGEDA operon apparently requires the presence of associated ribosome binding sites.

Base Sequence↗

PubMed1982

Nucleotide sequence of the ilvB promoter-regulatory region: a biosynthetic operon controlled by attenuation and cyclic AMP.

The DNA sequence of the promoter-regulatory region of the ilvB operon of Escherichia coli was determined. This region encodes a potential leader polypeptide containing 32 amino acids, 12 of which are the regulatory amino acids valine and leucine. Approximately 50 residues downstream from the coding region for the potential leader peptide is a site for terminating transcription. In vitro transcription experiments show that transcription terminates at this site and produces a leader mRNA of approximately equal to 188 nucleotides. A model for the multivalent regulation of this operon by valyl- and leucyl-tRNA is proposed on the basis of the mutually exclusive formation of five strong stem-and-loop structures in the leader mRNA. In addition, the -35 and -70 regions of this sequence show close structural homologies to areas in cyclic AMP receptor protein (CRP)-dependent promoters reported to be important for CRP function. In vitro transcription from the ilvB promoter was greatly increased by cyclic AMP and CRP. Taken together, these data strongly suggest that the ilvB biosynthetic operon is negatively controlled by multivalent transcription termination and is positively regulated by cyclic AMP and CRP.

Base Sequence↗

PubMed1982

Overlap between ampC and frd operons on the Escherichia coli chromosome.

The promoter for the Escherichia coli ampC beta-lactamase gene is shown to be located within the last gene of the fumarate reductase (frd) operon. Evidence is presented that the ampC attenuator serves as the terminator for transcription of this preceding operon. The nucleotide sequence was determined for two proteins that were shown to be encoded by a DNA segment preceding the ampC gene. The two proteins consisted of 131 and 119 triplets and had molecular weights of 15,000 and 13,100, respectively. The twelve COOH-terminal amino acids of the 13,100 molecular weight protein were found to overlap the ampC promoter. Accordingly, a G.C insertion in the promoter gave both increased transcription of ampC and a frameshift in this overlapping gene, resulting in readthrough proteins. Thus, we describe a type of very compact genetic organization of operons in prokaryotes.

Base Sequence↗

PubMed1983

Promoter mutations that allow nifA-independent expression of the nitrogen fixation nifHDKY operon.

The nifHDKY operon of Klebsiella pneumoniae encodes for structural polypeptides of nitrogenase and requires the nifA gene product for transcription. Mutations that allow transcription of the nifHDKY operon in absence of the nifA gene product were characterized in plasmids containing the regulatory region of nifHDKY and nifH fused in phase to lacZ. beta-Galactosidase activity served as a measure for nifH expression. Most mutations were located in the nif regulatory region and included insertion sequence 2 (IS2) insertions, a sequence duplication, and a base substitution. In Escherichia coli, beta-galactosidase activity expressed from the mutant plasmids in the absence of nifA was 6-30% of the nifA-activated, parental level. Expression from most mutant plasmids was further increased by nifA. In K. pneumoniae, IS2-containing plasmids expressed low levels of beta-galactosidase and responded poorly, if at all, to activation by nifA, whereas expression from other mutant types was similar to that observed in E. coli. Nucleotide sequence analysis of two mutants indicated that sequences within 41 base pairs upstream to the nifH coding sequence were involved in nif-specific regulation. The results suggest that an inverted repeat element in this region, which could theoretically form a cruciform structure in the DNA, is involved in the transcriptional control of the nifHDKY operon.

Bacterial Proteins↗

PubMed1983

Attenuation control of pyrBI operon expression in Escherichia coli K-12.

The pyrBI operon of Escherichia coli K-12 encodes the subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2). Expression of this operon apparently is negatively regulated by the intracellular levels of UTP. To elucidate the regulatory mechanism in which UTP functions, the nucleotide sequence of the promoter-regulatory region of the pyrBI operon was determined and DNA fragments containing this region were transcribed in vitro. These experiments revealed a rho-independent transcriptional terminator (attenuator) located only 23 base pairs before the promoter-proximal end of the structural genes. Transcription initiated upstream at either of two potential pyrBI promoters was efficiently (approximately equal to 98%) terminated at this site, indicating that the regulation of pyrBI expression involves attenuation control. Additional features identified suggest a model for regulation in which the relative rates of UTP-dependent transcription within the pyrBI leader region and coupled translation of the leader transcript control transcriptional termination at the attenuator.

Amino Acid Sequence↗

PubMed1984

An operator at -280 base pairs that is required for repression of araBAD operon promoter: addition of DNA helical turns between the operator and promoter cyclically hinders repression.

A site has been found that is required for repression of the Escherichia coli araBAD operon. This site was detected by the in vivo properties of deletion mutants. In vitro protection studies with DNase I and dimethylsulfate showed that araC protein can specifically bind in this area to nucleotides lying at position -265 to -294 with respect to the araBAD operon promoter (PBAD) transcription start point. The previously known sites of protein binding in the ara operon lie between +20 and -160. Since the properties of deletion strains show that all the sites required for araBAD induction lie between +20 and -110, the new site at -280 exerts its repressive action over an unusually large distance along the DNA. Insertions of -16, -8, 0, 5, 11, 15, 24, and 31 base pairs of DNA between the new site and PBAD were constructed. Repression was impaired in those cases in which half-integral turns of the DNA helix were introduced, but repression was nearly normal for the insertions of 0, +11, and +31 base pairs.

AraC Transcription Factor↗

PubMed1984

Nucleotide sequence surrounding transcription initiation site of xylABC operon on TOL plasmid of Pseudomonas putida.

The xylABC operon on the TOL plasmid directs the synthesis of enzymes for conversion of toluene to benzoate and is positively controlled by the regulatory gene xylR. In the study here the nucleotide sequence was determined for the regulatory region of this operon. The in vivo transcription initiation site of the operon was determined by S1 nuclease and reverse transcriptase mapping. RNA was prepared from m-methylbenzyl alcohol-induced cells of Pseudomonas putida and Escherichia coli carrying pTN2, a derivative of the TOL plasmid containing the structural and regulatory genes of the entire toluene-degrading pathway. The amount of E. coli mRNA was estimated to be only 10% of that of P. putida mRNA. Consensus sequences of the -10 region (Pribnow box) and the -35 region (RNA polymerase recognition site) in E. coli genes were not found in the region preceding the transcription initiation site, whereas a sequence complementary to the 3' end of the 16S rRNA of Pseudomonas aeruginosa and E. coli existed in front of the predicted start codon of the xylA gene. These results explain the inefficient expression of TOL genes in E. coli.

Base Sequence↗

PubMed1985

Structural analysis of the umu operon required for inducible mutagenesis in Escherichia coli.

The nucleotide sequence of a 2.8-kilobase fragment of the Escherichia coli chromosome containing the umuDC genes has been determined. The DNA sequence specifies two open reading frames of 417 and 1266 nucleotides encoding proteins with calculated molecular weights of 15,063 and 47,677, respectively. From these and the previous results of genetic and biochemical studies on the cloned genes, we conclude that the former is the umuD and the latter is the umuC gene. In vitro transcription of the regulatory region of the umu operon revealed that (i) upstream of the coding region there is a promoter-operator complex having the consensus sequence, CTGTATATAAAAACAG, of an SOS box, (ii) transcription of the umu operon begins at an adenine residue in the SOS box, and (iii) the LexA protein binds to the operator region, with an equilibrium dissociation constant (Kd) of 0.2, thereby repressing the transcription of the umuDC genes. These results indicate that the expression of the umu operon is under the coordinated control of the recA lexA gene products.

Amino Acid Sequence↗