Carabolite repression of the lac operon. Repression of translation.
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ABSTRACT:
SUBMITTER: Yudkin MD
PROVIDER: S-EPMC1184650 | biostudies-other | 1969 Jun
REPOSITORIES: biostudies-other
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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. 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. 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. Experiments were devised to show whether the point mutations L8 and L29 in the lac promoter alleviate transient repression. 2. Several recombinants were picked from matings between a single F(-)p(+) strain and Hfr strains carrying mutations L8 and L29. All of the 19 p(-) recombinants tested proved to suffer no transient repression, whereas all of the eight p(+) recombinants tested suffered prolonged transient repression. 3. A diploid strain was constructed in which more than 90% of the thiogalactoside transacetylase is synthesized from the episome with a wild-type lac promoter, whereas 100% of the beta-galactosidase is synthesized from the chromosome with a promoter carrying mutation L8. In this diploid the synthesis of thiogalactoside transacetylase suffered transient repression but the synthesis of beta-galactosidase did not. 4. Exactly similar results were obtained with a diploid strain in which the chromosomal promoter carried mutation L29. 5. The same diploid strains were used in experiments to show whether mutations L8 and L29 alleviate the severe catabolite repression caused by growth in glucose plus gluconate. In both strains glucose+gluconate repressed the synthesis of beta-galactosidase much less than the synthesis of thiogalactoside transacetylase. 6. These and previously reported results can be explained by assuming (a) that both mutations L8 and L29 render the lac promoter partially, but not completely, insensitive to catabolite repression, and (b) that transient repression is an exceptionally severe form of catabolite repression.
| S-EPMC1176997 | biostudies-other
Project description:BackgroundClassical bacteriological characteristics of Salmonella enterica indicate that the members of this species are unable to utilize lactose as a carbon source. However, lactose-fermenting (Lac+) strains of several Salmonella serovars have been isolated from different foodborne outbreaks as well as different geographical regions worldwide. In the present study, we sequenced the genomes of 13 Lac + S. enterica isolates and characterized the lac region, comparing it to the lac region in other enteric bacterial species.ResultsGenetic analysis of the lac operons in the S. enterica genomes revealed that they all contain intact lacI, lacZ, and lacY genes. However, lacA was truncated in all of the S. enterica subsp. enterica isolates, encoding a 56 amino acid peptide rather than the full length 220 amino acid LacA protein. Molecular analyses of the 13 isolates revealed that the lac operon resided on a plasmid in some strains and in others was integrated into the bacterial chromosome. In most cases, an insertion sequence flanked at least one end of the operon. Interestingly, the S. enterica Montevideo and S. enterica Senftenberg isolates were found to harbor a plasmid with a high degree of sequence similarity to a plasmid from Klebsiella pneumoniae strain NK29 that also harbors the lac operon. In addition, two S. enterica Tennessee isolates carried two copies of the lac operon. Phylogenetic analysis based on lacIZY gene sequences determines distinct clusters, and reveals a greater correlation between lacIZY sequence and flanking organization than with either bacterial species or genomic location.ConclusionsOur results indicate that the lac region is highly mobile among Enterobacteriaceae and demonstrate that the Lac + S. enterica subsp. enterica serovars acquired the lac region through parallel events. The acquisition of the lac operon by several S. enterica serovars may be indicative of environmental adaptation by these bacteria.
| S-EPMC4549013 | biostudies-literature
Project description:Concept inventories (CIs) are valuable tools for educators that assess student achievement and identify misconceptions held by students. Results of student responses can be used to adjust or develop new instructional methods for a given topic. The regulation of gene expression in both prokaryotes and eukaryotes is an important concept in genetics and one that is particularly challenging for undergraduate students. As part of a larger study examining instructional methods related to gene regulation, the authors developed a 12-item CI assessing student knowledge of the lac operon. Using an established protocol, the authors wrote open-ended questions and conducted in-class testing with undergraduate microbiology and genetics students to discover common errors made by students about the lac operon and to determine aspects of item validity. Using these results, we constructed a 12-item multiple-choice lac operon CI called the Lac Operon Concept Inventory (LOCI), The LOCI was reviewed by two experts in the field for content validity. The LOCI underwent item analysis and was assessed for reliability with a sample of undergraduate genetics students (n = 115). The data obtained were found to be valid and reliable (coefficient alpha = 0.994) with adequate discriminatory power and item difficulty.
| S-EPMC4909346 | biostudies-literature
Project description:The inflexibility of double-stranded DNA with respect to bending and twisting is well established in vitro. Understanding apparent DNA physical properties in vivo is a greater challenge. Here, we exploit repression looping with components of the Escherichia coli lac operon to monitor DNA flexibility in living cells. We create a minimal system for testing the shortest possible DNA repression loops that contain an E. coli promoter, and compare the results to prior experiments. Our data reveal that loop-independent repression occurs for certain tight operator/promoter spacings. When only loop-dependent repression is considered, fits to a thermodynamic model show that DNA twisting limits looping in vivo, although the apparent DNA twist flexibility is 2- to 4-fold higher than in vitro. In contrast, length-dependent resistance to DNA bending is not observed in these experiments, even for the shortest loops constraining <0.4 persistence lengths of DNA. As observed previously for other looping configurations, loss of the nucleoid protein heat unstable (HU) markedly disables DNA looping in vivo. Length-independent DNA bending energy may reflect the activities of architectural proteins and the structure of the DNA topological domain. We suggest that the shortest loops are formed in apical loops rather than along the DNA plectonemic superhelix.
| S-EPMC3001091 | biostudies-literature
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:Populations of Escherichia coli selected in constant and fluctuating environments containing lactose often adapt by substituting mutations in the lacI repressor that cause constitutive expression of the lac operon. These mutations occur at a high rate and provide a significant benefit. Despite this, eight of 24 populations evolved for 8,000 generations in environments containing lactose contained no detectable repressor mutations. We report here on the basis of this observation. We find that, given relevant mutation rates, repressor mutations are expected to have fixed in all evolved populations if they had maintained the same fitness effect they confer when introduced to the ancestor. In fact, reconstruction experiments demonstrate that repressor mutations have become neutral or deleterious in those populations in which they were not detectable. Populations not fixing repressor mutations nevertheless reached the same fitness as those that did fix them, indicating that they followed an alternative evolutionary path that made redundant the potential benefit of the repressor mutation, but involved unique mutations of equivalent benefit. We identify a mutation occurring in the promoter region of the uspB gene as a candidate for influencing the selective choice between these paths. Our results detail an example of historical contingency leading to divergent evolutionary outcomes.
| S-EPMC8233506 | biostudies-literature
Project description:The role of stochasticity on gene expression is widely discussed. Both potential advantages and disadvantages have been revealed. In some systems, noise in gene expression has been quantified, in among others the lac operon of Escherichia coli. Whether stochastic gene expression in this system is detrimental or beneficial for the cells is, however, still unclear. We are interested in the effects of stochasticity from an evolutionary point of view. We study this question in the lac operon, taking a computational approach: using a detailed, quantitative, spatial model, we evolve through a mutation-selection process the shape of the promoter function and therewith the effective amount of stochasticity. We find that noise values for lactose, the natural inducer, are much lower than for artificial, nonmetabolizable inducers, because these artificial inducers experience a stronger positive feedback. In the evolved promoter functions, noise due to stochasticity in gene expression, when induced by lactose, only plays a very minor role in short-term physiological adaptation, because other sources of population heterogeneity dominate. Finally, promoter functions evolved in the stochastic model evolve to higher repressed transcription rates than those evolved in a deterministic version of the model. This causes these promoter functions to experience less stochasticity in gene expression. We show that a high repression rate and hence high stochasticity increases the delay in lactose uptake in a variable environment. We conclude that the lac operon evolved such that the impact of stochastic gene expression is minor in its natural environment, but happens to respond with much stronger stochasticity when confronted with artificial inducers. In this particular system, we have shown that stochasticity is detrimental. Moreover, we demonstrate that in silico evolution in a quantitative model, by mutating the parameters of interest, is a promising way to unravel the functional properties of biological systems.
| S-EPMC1894820 | biostudies-literature