Effect of point mutations in the lac promoter on transient and severe catabolite repression of the lac operon of Escherichia coli.
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ABSTRACT:
SUBMITTER: Yudkin MD
PROVIDER: S-EPMC1176997 | biostudies-other | 1971 Jul
REPOSITORIES: biostudies-other
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Project description:1. Several lac diploid strains of Escherichia coli were constructed and tested to discover whether mutations in the lac promoter alleviate catabolite repression. 2. In each of these diploids the chromosome carries one of the promoter mutations, L8, L29 or L1; so that the rate of synthesis of the enzymes of the lac operon is only 2-6% of the fully induced wild-type. Each diploid harbours the episome F'lacM15 that specifies the synthesis of thiogalactoside transacetylase under the control of intact regulator, promoter and operator regions, but has a deletion in the structural gene for beta-galactosidase. In each diploid more than 90% of the thiogalactoside transacetylase is synthesized from the episome, and 100% of the beta-galactosidase is synthesized from the chromosome, and comparison of the extent of catabolite repression that the two enzymes suffered indicated whether the chromosomal promoter mutation relieves catabolite repression. 3. In the strains in which the promoter carries either of the point mutations L8 or L29 the enzymes were equally repressed, suggesting that neither L8 nor L29 affects catabolite repression. 4. In a diploid strain harbouring the same episome but carrying deletion L1 on the chromosome, synthesis of beta-galactosidase suffered much less repression than that of thiogalactoside transacetylase. 5. In a diploid strain in which the chromosome carries L1 and also a second mutation that increases the rate of expression of lac to that permitted by L8 or L29, the synthesis of beta-galactosidase again suffered much less repression than the synthesis of thiogalactoside transacetylase. 6. The effect of L1 (which deletes the boundary between the i gene and the lac promoter) is ascribed to its bringing the expression of lac under the control of the promoter of the i gene. 7. Even in strains carrying L1, some catabolite repression persists; this is not due to a trans effect from the episome since it occurs equally in a haploid strain with L1.
| S-EPMC1179282 | biostudies-other
Project description:1. Experiments were carried out to distinguish the contributions of transcriptional and translational repression to catabolite repression of the lac operon. 2. In strain EZ16-3-G of Escherichia coli the synthesis of thiogalactoside transacetylase is directed by a gene situated on an episome, and the operator, promotor and regulator genes that lay cis to this gene have been deleted, so that the normal mechanism for controlling transcription is abolished. The extent of catabolite repression in this strain was much less than that in wild-type strains. 3. The same episome is responsible for the synthesis of thiogalactoside transacetylase in strain RM32/F'd25, and in this strain a second lac operon directs the synthesis of beta-galactosidase under the control of a wild-type operator-promotor-regulator system. The extent of catabolite repression of thiogalactoside transacetylase in strain RM32/F'd25 was substantially more than in strain EZ16-3-G, but less than that of beta-galactosidase in strain RM32/F'd25. 4. Since the synthesis of thiogalactoside transacetylase in these organisms is presumably subject to translational repression only, it is concluded that in strain RM32/F'd25 the synthesis of beta-galactosidase is subject to both transcriptional and translational repression. It is also concluded that the extent of translational repression varies between strains.
| S-EPMC1184856 | biostudies-other
Project description:1. Catabolite repression of beta-galactosidase and of thiogalactoside transacetylase was studied in several strains of Escherichia coli K 12, in an attempt to show whether a single site within the structural genes of the lac operon co-ordinately controls translational repression for the two enzymes. In all experiments the rate of synthesis of the enzymes was compared in glycerol-minimal medium and in glucose-minimal medium. 2. In a wild-type strain, glucose repressed the synthesis of the two enzymes equally. 3. The possibility that repression was co-ordinate was investigated by studies of mutant strains that carry deletions in the genes for beta-galactosidase or galactoside permease or both. In all of the strains with deletions, the repression of thiogalactoside transacetylase persisted, and it is concluded that there is no part of the structural gene for beta-galactosidase that is essential for catabolite repression of thiogalactoside transacetylase. 4. Subculture of one strain through several transfers in rich medium greatly increased its susceptibility to catabolite repression by glucose. It is concluded that unknown features of the genotype can markedly affect sensitivity to catabolite repression. 5. These results make it clear that one cannot draw valid conclusions about the effect of known genotypic differences on catabolite repression from a comparison of two separate strains; to study the effect of a particular genetic change in a lac operon it is necessary to construct a partially diploid strain so that catabolite repression suffered by one lac operon can be compared with that suffered by another. 6. Four such partial diploids were constructed. In all of them catabolite repression of beta-galactosidase synthesized by one operon was equal in extent to catabolite repression of thiogalactoside transacetylase synthesized by the other. 7. Taken together, these results suggest that catabolite repression of beta-galactosidase and thiogalactoside transacetylase is separate but equal.
| S-EPMC1184857 | biostudies-other
Project description:1. Acute transient catabolite repression of beta-galactosidase synthesis, observed when glucose is added to glycerol-grown cells of Escherichia coli (Moses & Prevost, 1966), requires the presence of a functional operator gene (o) in the lactose operon. Total deletion of the operator gene abolished acute transient repression, even in the presence of a functional regulator gene (i). 2. Regulator constitutives (i(-)) also show transient repression provided that the operator gene is functional. Regulator deletion mutants (i(del)), with which to test specifically the role of the i gene, have not so far been available. 3. The above mutants, showing various changes in the lactose operon, show no alteration in the effect of glucose on induced tryptophanase synthesis. Glucose metabolism, as measured in terms of the release of (14)CO(2) from [1-(14)C]glucose and [6-(14)C]glucose, also showed no differences between strains exhibiting or not exhibiting transient repression. This suggests no change in the operation of the pentose phosphate cycle, a metabolic activity known to be of paramount importance for glucose repression of beta-galactosidase synthesis (Prevost & Moses, 1967). 4. Chronic permanent repression by glucose of beta-galactosidase synthesis (less severe in degree than acute transient repression) persists in strains in which transient repression has been genetically abolished. Constitutive alkaline-phosphatase synthesis, which shows no transient repression, also demonstrates chronic permanent repression by glucose. 5. Chloramphenicol repression also persists in mutants with no transient repression, and also affects alkaline phosphatase. It is suggested that chronic permanent repression and chloramphenicol repression are non-specific, and that they do not influence beta-galactosidase synthesis via the regulatory system of the lactose operon.
| S-EPMC1270416 | biostudies-other
Project description:1. Experiments were devised to show whether catabolite repression of beta-galactosidase synthesis operates at the level of transcription or of translation. Escherichia coli K12 was induced for a short period in non-repressing medium (glycerol-minimal medium), and transcription of the lac operon was terminated by either of two methods; glucose was then added as a source of the catabolite repressor during the subsequent translation of the accumulated beta-galactosidase messenger RNA. 2. When induced bacteria in glycerol medium were infected with T6 phage, which is known to halt transcription, the addition of glucose up to 3min. later diminished the yield of beta-galactosidase. 3. When induced bacteria in glycerol medium were removed from the inducer and resuspended in fresh medium (a process that is also known to halt transcription), the yield of enzyme was again diminished by the presence of glucose in the resuspension medium. 4. It is concluded that repression of beta-galactosidase synthesis can be brought about by the presence of glucose during the translation phase only. 5. In E. coli strain 300U the effect on translation was sufficient to account for almost all the catabolite repression of beta-galactosidase synthesis observed during exponential growth of the organism in glucose-minimal medium. In E. coli strain 200P, however, much more severe repression occurred during exponential growth, and an additional effect of glucose is postulated.
| S-EPMC1184650 | biostudies-other
Project description:The Escherichia coli lactose (lac) operon encodes the first genetic switch to be discovered, and lac remains a paradigm for studying negative and positive control of gene expression. Negative control is believed to involve competition of RNA polymerase and Lac repressor for overlapping binding sites. Contributions to the local Lac repressor concentration come from free repressor and repressor delivered to the operator from remote auxiliary operators by DNA looping. Long-standing questions persist concerning the actual role of DNA looping in the mechanism of promoter repression. Here, we use experiments in living bacteria to resolve four of these questions. We show that the distance dependence of repression enhancement is comparable for upstream and downstream auxiliary operators, confirming the hypothesis that repressor concentration increase is the principal mechanism of repression loops. We find that as few as four turns of DNA can be constrained in a stable loop by Lac repressor. We show that RNA polymerase is not trapped at repressed promoters. Finally, we show that constraining a promoter in a tight DNA loop is sufficient for repression even when promoter and operator do not overlap.
| S-EPMC3592455 | biostudies-literature
Project description:The formation of glycerol-3-phosphate (G3P) in cells growing on TB causes catabolite repression, as shown by the reduction in malT expression. For this repression to occur, the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular EIIA(Glc), as well as the adenylate cyclase and the cyclic AMP-catabolite activator protein system, have to be present. We followed the level of EIIA(Glc) phosphorylation after the addition of glycerol or G3P. In contrast to glucose, which causes a dramatic shift to the dephosphorylated form, glycerol or G3P only slightly increased the amount of dephosphorylated EIIA(Glc). Isopropyl-beta-D-thiogalactopyranoside-induced overexpression of EIIA(Glc) did not prevent repression by G3P, excluding the possibility that G3P-mediated catabolite repression is due to the formation of unphosphorylated EIIA(Glc). A mutant carrying a C-terminally truncated adenylate cyclase was no longer subject to G3P-mediated repression. We conclude that the stimulation of adenylate cyclase by phosphorylated EIIA(Glc) is controlled by G3P and other phosphorylated sugars such as D-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds. Further metabolism of these compounds is not necessary for repression. Two-dimensional polyacrylamide gel electrophoresis was used to obtain an overview of proteins that are subject to catabolite repression by glycerol. Some of the prominently repressed proteins were identified by peptide mass fingerprinting. Among these were periplasmic binding proteins (glutamine and oligopeptide binding protein, for example), enzymes of the tricarboxylic acid cycle, aldehyde dehydrogenase, Dps (a stress-induced DNA binding protein), and D-tagatose-1,6-bisphosphate aldolase.
| S-EPMC135075 | biostudies-literature
Project description:Chromatin immunoprecipitation, followed by quantification of immunoprecipitated DNA, can be used to measure RNA polymerase binding to any DNA segment in Escherichia coli By calibrating measurements against the signal from a single RNA polymerase bound at a single promoter, we can calculate both promoter occupancy levels and the flux of transcribing RNA polymerase through transcription units. Here, we have applied the methodology to the E. coli lactose operon promoter. We confirm that promoter occupancy is limited by recruitment and that the supply of RNA polymerase to the lactose operon promoter depends on its location in the E. coli chromosome. Measurements of RNA polymerase binding to DNA segments within the lactose operon show that flux of RNA polymerase through the operon is low, with, on average, over 18 s elapsing between the passage of transcribing polymerases. Similar low levels of flux were found when semi-synthetic promoters were used to drive transcript initiation, even when the promoter elements were changed to ensure full occupancy of the promoter by RNA polymerase.This article is part of the themed issue 'The new bacteriology'.
| S-EPMC5052750 | biostudies-literature
Project description:Bacterial biofilms are complex communities of cells containing an increased prevalence of dormant cells known as persisters, which are characterized by an up-regulation of genes known as toxin-antitoxin (TA) modules. The association of toxins with their cognate antitoxins neutralizes toxin activity, allowing for normal cell growth. Additionally, protein antitoxins bind their own promoters and repress transcription, whereas the toxins serve as co-repressors. Recently, TA pairs have been shown to regulate their own transcription through a phenomenon known as conditional cooperativity, where the TA complexes bind operator DNA and repress transcription only when present in the proper stoichiometric amounts. The most differentially up-regulated gene in persister cells is mqsR, a gene that, with the antitoxin mqsA, constitutes a TA module. Here, we reveal that, unlike other TA systems, MqsR is not a transcription co-repressor but instead functions to destabilize the MqsA-DNA complex. We further show that DNA binding is not regulated by conditional cooperativity. Finally, using biophysical studies, we show that complex formation between MqsR and MqsA results in an exceptionally stable interaction, resulting in a subnanomolar dissociation constant that is similar to that observed between MqsA and DNA. In combination with crystallographic studies, this work reveals that MqsA binding to DNA and MqsR is mutually exclusive. To our knowledge, this is the first TA system in which the toxin does not function as a transcriptional co-repressor, but instead functions to destabilize the antitoxin-operator complex under all conditions, and thus defines another unique feature of the mqsRA TA module.
| S-EPMC3543012 | biostudies-literature
Project description:1. Repression by glucose of beta-galactosidase synthesis is spontaneously reversible in all strains of Escherichia coli examined long before the glucose has all been consumed. The extent of recovery and the time necessary for reversal differ among various strains. Other inducible enzymes show similar effects. 2. This transient effect of glucose repression is observed in constitutive (i(-)) and permease-less (y(-)) cells as well as in the corresponding i(+) and y(+) strains. 3. Repression is exerted by several rapidly metabolizable substrates (galactose, ribose and ribonucleosides) but not by non-metabolized or poorly metabolized compounds (2-deoxyglucose, 2-deoxyribose, phenyl thio-beta-galactoside and 2-deoxyribonucleosides). 4. The transient repression with glucose is observed in inducible cells supplied with a powerful inducer of beta-galactosidase synthesis (e.g. isopropyl thio-beta-galactoside) but not with a weak inducer (lactose); in the latter instance glucose repression is permanent. Diauxic growth on glucose plus lactose can be abolished by including isopropyl thio-beta-galactoside in the medium. 5. In some strains phosphate starvation increases catabolite repression; in others it relieves it. Adenine starvation in an adenine-requiring mutant also relieves catabolite repression by glycerol but not that by glucose. Restoration of phosphate or adenine to cells starved of these nutrients causes a pronounced temporary repression. Alkaline-phosphatase synthesis is not affected by the availability of adenine. 6. During periods of transient repression of induced enzyme synthesis the differential rate of RNA synthesis, measured by labelled uracil incorporation in 2min. pulses, shows a temporary rise. 7. The differential rate of uracil incorporation into RNA falls during exponential growth of batch cultures of E. coli. This is equally true for uracil-requiring and non-requiring strains. The fall in the rate of incorporation has been shown to be due to a real fall in the rate of RNA synthesis. The significance of the changes in the rate of RNA synthesis is discussed. 8. A partial model of catabolite repression is presented with suggestions for determining the chemical identification of the catabolite co-repressor itself.
| S-EPMC1265142 | biostudies-other