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Current Opinion in Structural Biology Feb 1997For many years the lac operon of Escherichia coli has been the paradigm for gene regulation. Recently, the structures of the lac repressor core bound to... (Review)
Review
For many years the lac operon of Escherichia coli has been the paradigm for gene regulation. Recently, the structures of the lac repressor core bound to isopropyl-beta-D-1-thiogalactoside (IPTG), the intact apo lac repressor, the intact lac repressor complexes with IPTG and a 21-base-pair symmetric operator, and the refined headpiece of the repressor have been determined. These structures have provided a framework for understanding a wealth of biochemical and genetic information. An analysis of these structures, as well as a description of their function and a comparison to homologous proteins, is now possible.
Topics: Allosteric Regulation; Bacterial Proteins; Binding Sites; Escherichia coli Proteins; Lac Operon; Lac Repressors; Protein Binding; Repressor Proteins
PubMed: 9032054
DOI: 10.1016/s0959-440x(97)80010-3 -
Comptes Rendus Biologies Jun 2005Transcription of E. coli lac operon by RNA polymerase (RNAP) is a classic example of how the basic functions of this enzyme, specifically the ability to recognize/bind... (Review)
Review
Transcription of E. coli lac operon by RNA polymerase (RNAP) is a classic example of how the basic functions of this enzyme, specifically the ability to recognize/bind promoters, melt the DNA and initiate RNA synthesis, is positively regulated by transcription activators, such as cyclic AMP-receptor protein, CRP, and negatively regulated by lac-repressor, LacI. In this review, we discuss the recent progress in structural and biochemical studies of RNAP and its binary and ternary complexes with CRP and lac promoter. With structural information now available for RNAP and models of binary and ternary elongation complexes, the interaction between these factors and RNAP can be modeled, and possible molecular mechanisms of their action can be inferred.
Topics: Cyclic AMP Receptor Protein; DNA; DNA-Directed RNA Polymerases; Escherichia coli; Lac Operon; Models, Molecular; Promoter Regions, Genetic; Protein Conformation; Transcription, Genetic
PubMed: 15950164
DOI: 10.1016/j.crvi.2005.03.007 -
Mathematical Biosciences May 2011The lactose (lac) operon of Escherichia coli serves as the paradigm for gene regulation, not only for bacteria, but also for all biological systems from simple phage to... (Review)
Review
The lactose (lac) operon of Escherichia coli serves as the paradigm for gene regulation, not only for bacteria, but also for all biological systems from simple phage to humans. The details of the systems may differ, but the key conceptual framework remains, and the original system continues to reveal deeper insights with continued experimental and theoretical study. Nearly as long lasting in impact as the pivotal work of Jacob and Monod is the classic experiment of Novick and Weiner in which they demonstrated all-or-none gene expression in response to an artificial inducer. These results are often cited in claims that normal gene expression is in fact a discontinuous bistable phenomenon. In this paper, I review several levels of analysis of the lac system and introduce another perspective based on the construction of the system design space. These represent variations on a theme, based on a simply stated design principle, that captures the key qualitative features of the system in a largely mechanism-independent fashion. Moreover, this principle can be readily interpreted in terms of specific mechanisms to make predictions regarding monostable vs. bistable behavior. The regions of design space representing bifurcations are compared with the corresponding regions identified through bifurcation analysis. I present evidence based on biological considerations as well as modeling and analysis to suggest that induction of the lac system in its natural setting is a monostable continuously graded phenomenon. Nevertheless, it must be acknowledged that the lac stability question remains unsettled, and it undoubtedly will remain so until there are definitive experimental results.
Topics: Algorithms; Gene Expression Regulation; Humans; Lac Operon; Models, Genetic; Systems Theory
PubMed: 21414326
DOI: 10.1016/j.mbs.2011.03.008 -
Biophysical Journal Aug 2019Mechanistic models of stochastic gene expression are of considerable interest, but their complexity often precludes tractable analytical expressions for messenger RNA...
Mechanistic models of stochastic gene expression are of considerable interest, but their complexity often precludes tractable analytical expressions for messenger RNA (mRNA) and protein distributions. The lac operon of Escherichia coli is a model system with regulatory elements such as multiple operators and DNA looping that are shared by many operons. Although this system is complex, intuition suggests that fast DNA looping may simplify it by causing the repressor-bound states of the operon to equilibrate rapidly, thus ensuring that the subsequent dynamics are governed by slow transitions between the repressor-free and the equilibrated repressor-bound states. Here, we show that this intuition is correct by applying singular perturbation theory to a mechanistic model of lac transcription with the scaled time constant of DNA looping as the perturbation parameter. We find that at steady state, the repressor-bound states satisfy detailed balance and are dominated by the looped states; moreover, the interaction between the repressor-free and the equilibrated repressor-bound states is described by an extension of the Peccoud-Ycart two-state model in which both (repressor-free and repressor-bound) states support transcription. The solution of this extended two-state model reveals that the steady-state mRNA distribution is a mixture of the Poisson and negative hypergeometric distributions, which reflects mRNAs obtained by transcription from the repressor-bound and repressor-free states. Finally, we show that the physics revealed by perturbation theory makes it easy to derive the extended two-state model equations for complex regulatory architectures.
Topics: Escherichia coli; Gene Expression Regulation, Bacterial; Lac Operon; Models, Genetic; RNA, Messenger; Single-Cell Analysis
PubMed: 31331635
DOI: 10.1016/j.bpj.2019.06.029 -
Molecular Microbiology Jan 2021When Streptococcus mutans is transferred from a preferred carbohydrate (glucose or fructose) to lactose, initiation of growth can take several hours, and substantial...
When Streptococcus mutans is transferred from a preferred carbohydrate (glucose or fructose) to lactose, initiation of growth can take several hours, and substantial amounts of glucose are released during growth. Here, S. mutans strains UA159 and GS-5 were examined for stochastic behaviors in transcription of the lac operon. Using a gfp reporter fusion, we demonstrated that induction of the lac operon occurs in only a fraction of the population, with prior exposure to carbohydrate source and strain influencing the magniture of the sub-population response. Lower glucokinase activity in GS-5 was associated with release of substantially more glucose than UA159 and significantly lower lac expression. Mutants unable to use lactose grew on lactose as the sole carbohydrate when strains with an intact lac operon were also present in the cultures, indicative of the potential for population cheating. Utilizing a set of engineered obligate cheating and non-cheating strains, we confirmed that cheating can sustain a heterogeneous population. Futher, obligate cheaters of GS-5 competed well with the non-cheaters and showed a high degree of competitive fitness in a human-derived consortium biofilm model. The results show that bet-hedging behaviors in carbohydrate metabolism may substantially influence the composition and pathogenic potential of oral biofilms.
Topics: Biofilms; Carbohydrate Metabolism; Fructose; Gene Expression; Gene Expression Regulation, Bacterial; Glucose; Lac Operon; Lactose; Operon; Streptococcus mutans
PubMed: 32881164
DOI: 10.1111/mmi.14596 -
Molecular Systems Biology 2006The formation and regulation of macromolecular complexes provides the backbone of most cellular processes, including gene regulation and signal transduction. The... (Review)
Review
The formation and regulation of macromolecular complexes provides the backbone of most cellular processes, including gene regulation and signal transduction. The inherent complexity of assembling macromolecular structures makes current computational methods strongly limited for understanding how the physical interactions between cellular components give rise to systemic properties of cells. Here, we present a stochastic approach to study the dynamics of networks formed by macromolecular complexes in terms of the molecular interactions of their components. Exploiting key thermodynamic concepts, this approach makes it possible to both estimate reaction rates and incorporate the resulting assembly dynamics into the stochastic kinetics of cellular networks. As prototype systems, we consider the lac operon and phage lambda induction switches, which rely on the formation of DNA loops by proteins and on the integration of these protein-DNA complexes into intracellular networks. This cross-scale approach offers an effective starting point to move forward from network diagrams, such as those of protein-protein and DNA-protein interaction networks, to the actual dynamics of cellular processes.
Topics: Computational Biology; DNA; Lac Operon; Macromolecular Substances; Proteins; Stochastic Processes; Thermodynamics
PubMed: 16738569
DOI: 10.1038/msb4100061 -
CBE Life Sciences Education 2016Concept inventories (CIs) are valuable tools for educators that assess student achievement and identify misconceptions held by students. Results of student responses can...
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.
Topics: Educational Measurement; Female; Humans; Lac Operon; Male; Pilot Projects; Reproducibility of Results; Students; Thinking
PubMed: 27252300
DOI: 10.1187/cbe.15-07-0162 -
Journal of Bacteriology May 2017The (lactose) operon (which processes β-galactosides) and the (melibiose) operon (which processes α-galactosides) of have a close historical connection. A number of...
The (lactose) operon (which processes β-galactosides) and the (melibiose) operon (which processes α-galactosides) of have a close historical connection. A number of shared substrates and effectors of the permeases and regulatory proteins have been reported over the years. Until now, β-thiogalactosides like TMG (methyl-β-d-thiogalactopyranoside) and IPTG (isopropyl-β-d-thiogalactopyranoside) have not generally been considered to be inducers of the operon. The same is true for β-galactosides such as lactose [β-d-galactopyranosyl-(1→4)-d-glucose], which is a substrate but is not itself an inducer of the operon. This report shows that all three sugars can induce the operon significantly when they are accumulated in the cell by Lac permease. Strong induction by β-thiogalactosides is observed in the presence of Lac permease, and strong induction by lactose (more than 200-fold) is observed in the absence of β-galactosidase. This finding calls for reevaluation of TMG uptake experiments as assays for Lac permease that were performed with strains. The typical textbook picture of bacterial operons is that of stand-alone units of genetic information that perform, in a regulated manner, well-defined cellular functions. Less attention is given to the extensive interactions that can be found between operons. Well-described examples of such interactions are the effector molecules shared by the and operons. Here, we show that this set has to be extended to include β-galactosides, which have been, until now, considered not to effect the expression of the operon. That they can be inducers of the operon as well as the operon has not been noted in decades of research because of the genetic background used in previous studies.
Topics: Escherichia coli; Galactosides; Glucose; Lac Operon; Lactose; Melibiose; Membrane Transport Proteins; Operon; beta-Galactosidase
PubMed: 28193904
DOI: 10.1128/JB.00796-16 -
BMC Microbiology Aug 2015Classical bacteriological characteristics of Salmonella enterica indicate that the members of this species are unable to utilize lactose as a carbon source. However,...
BACKGROUND
Classical 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.
RESULTS
Genetic 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.
CONCLUSIONS
Our 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.
Topics: Chromosomes, Bacterial; Cluster Analysis; DNA Transposable Elements; DNA, Bacterial; Gene Order; Genome, Bacterial; Lac Operon; Molecular Sequence Data; Phylogeny; Plasmids; Salmonella enterica; Sequence Analysis, DNA; Sequence Homology
PubMed: 26303940
DOI: 10.1186/s12866-015-0511-8 -
ACS Synthetic Biology Feb 2023Reverse genetics (RG) systems have been instrumental for determining the molecular aspects of viral replication, pathogenesis, and for the development of therapeutics....
Reverse genetics (RG) systems have been instrumental for determining the molecular aspects of viral replication, pathogenesis, and for the development of therapeutics. Here, we demonstrate that genes encoding the influenza surface antigens hemagglutinin and neuraminidase have varying stability when cloned into a common RG plasmid and transformed into . Using GFP as a reporter, we demonstrate that expresses the target genes in the RG plasmid at low levels. Incorporating operators or a transcriptional terminator into the plasmid reduced expression and stabilized the viral genes to varying degrees. Sandwiching the viral gene between two operators provided the largest contribution to stability and we confirmed the stabilization is Lac repressor-dependent and crucial for subsequent plasmid propagations in . Viruses rescued from the operator-stabilized plasmid displayed similar kinetics and titers to the original plasmid in two different viral backbones. Together, these results indicate that silencing transcription from the plasmid in helps to maintain the correct influenza gene sequence and that the operator addition does not impair virus production. It is envisaged that sandwiching DNA segments between operators can be used for reducing DNA segment instability in any plasmid that is propagated in which express the Lac repressor.
Topics: Humans; Escherichia coli; Lac Repressors; Influenza, Human; Reverse Genetics; Plasmids; Lac Operon
PubMed: 36716395
DOI: 10.1021/acssynbio.2c00358