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Regulation of Cra protein level in E coli

Regulation of Cra protein level in E coli


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Catabolite Activator/Repressor, Cra protein (formerly known as Fructure Repressor FruR) plays a significant role in central carbon metabolism of E coli. Its activity is inhibited by fructose-1,6-bisphosphate (FBP); Cra up-regulate gluconeogeneis pathways and down-regulate glycolysis and carbon-uptake pathways. I found enough literature on how Cra plays a regulatory role in metabolism.

However, I am unable to find out explanation of one experimental observation: with increase in glucose uptake rate the transcript level of cra decreases. What is the mechanism for this. Further, are Cra proteins degraded as the glucose uptake rate increase, if so what is the regulatory mechanism?


Catabolite repressor/activator

<p>The annotation score provides a heuristic measure of the annotation content of a UniProtKB entry or proteome. This score <strong>cannot</strong> be used as a measure of the accuracy of the annotation as we cannot define the 'correct annotation' for any given protein.<p><a href='/help/annotation_score' target='_top'>More. </a></p> - Protein inferred from homology i <p>This indicates the type of evidence that supports the existence of the protein. Note that the 'protein existence' evidence does not give information on the accuracy or correctness of the sequence(s) displayed.<p><a href='/help/protein_existence' target='_top'>More. </a></p>

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Milton Saier

Our laboratory has three primary research interests, one concerned with transcriptional and metabolic regulation in bacteria, a second with transport protein evolution, and a third with the recently identified process of transposon-mediated directed mutation. We also maintain the IUBMB-approved Transporter Classification Database, TCDB, which classifies transport systems found in all living organisms on Earth into five categories: class, subclass, family, subfamily and transport system.

Our laboratory takes a multidisciplinary approach to science, using biochemical, molecular genetic, physiological, and computational approaches. Directed mutation is a proposed process that allows mutations to occur at higher frequencies when they are beneficial than when detrimental. Until recently, the existence of such a process has been controversial. However, we have described a novel mechanism of directed mutation mediated by the transposon, IS 5 in Escherichia coli. crp deletion mutants mutate specifically to glycerol utilization (Glp+) at rates that are enhanced by glycerol or the loss of the glycerol repressor (GlpR), depressed by glucose or glpR overexpression, and RecA-independent. Of the four tandem GlpR binding sites ( O1–O4) upstream of the glpFK operon, O4 specifically controls glpFK expression while O1 primarily controls mutation rate in a process mediated by IS 5 hopping to a specific site on the E. coli chromosome upstream of the glpFK promoter. IS 5 insertion into other gene activation sites is unaffected by the presence of glycerol or the loss of GlpR. The results establish an example of transposon-mediated directed mutation, identify the protein responsible and define the mechanism involved. Most recently, we have identified two additional operons in E. coli that appear to be subject to transposon-mediated directed mutation: The flhDC flagellar master switch operon controlling motility and the fucAO operon controlling L-fucose and propanediol utilization.

Our efforts have revealed three basic mechanisms of transcriptional control concerned with catabolite repression/activation in bacteria. Two of these occur in E. coli, and one occurs in B. subtilis. In E. coli, two DNA binding proteins, the cyclic AMP receptor protein (Crp) and the catabolite repressor/activator (Cra) protein, mediate transcriptional regulation of hundreds of genes encoding key enzymes of carbon and energy metabolism. Virtually every pathway of carbon metabolism is subject to these regulatory constraints. Crp generally controls the initiation of exogenous carbon source metabolism and senses cytoplasmic cyclic AMP levels. These levels are controlled by complex mechanisms, involving phosphorylated proteins of the sugar-transporting phosphotransferase system (PTS). Cra generally controls the flux of carbon through metabolic pathways and senses cytoplasmic metabolite concentrations. Cra usually controls gene expression independently of Crp, but it sometimes acts cooperatively with or antagonistically to Crp, depending on the target gene. It thereby mediates catabolite repression of catabolic operons by an indirect mechanism. Most recently, we have demonstrated that Cra regulates growth by activating expression of the crp gene.

In B. subtilis and other Gram-positive bacteria, a metabolite-activated protein kinase phosphorylates a serine residue in a protein of the PTS called HPr. Phosphorylated HPr allosterically controls the activities of many target proteins (transport proteins, enzymes and transcription factors). It thereby controls the cytoplasmic concentrations of inducers as well as the activities of transcription factors that mediate catabolite repression. We are coming to realize that the mechanisms of catabolite control are very different for phylogenetically divergent bacteria.

Phylogenetic analyses of integral membrane transport protein sequences have yielded a plethora of information about the times of appearance, the routes of evolution, and the relative rates of divergence of the proteins and protein domains which comprise various families of transport systems. These studies have shown that families of transport proteins of similar topology have evolved independently of each other, at different times in evolutionary history, using different routes. They have also revealed extensive domain shuffling in some such families but not in others. The probable means by which energy coupling became superimposed on transport during the evolutionary process has also come to light.


Corepressors

As mentioned above, the synthesis of tryptophan from precursors available in the cell requires 5 enzymes. The genes encoding these are clustered together in a single operon with its own promoter and operator. In this case, however, the presence of tryptophan in the cell shuts down the operon. When Trp is present, it binds to a site on the Trp repressor and enables the Trp repressor to bind to the operator. When Trp is not present, the repressor leaves its operator, and transcription of the 5 enzyme-encoding genes begins.

Figure 9.1.3: Tryptophan Repressor courtesy of P. B. Sigler

The above picure shows stereo view of the tryptophan repressor (right side of each panel) bound to its operator DNA (left side). The repressor is a homodimer of two identical polypeptides (on either side of the horizontal red line). Binding to DNA occurs only when a molecule of tryptophan (red rings) is bound to each monomer of the repressor. The usefulness to the cell of this control mechanism is clear. The presence in the cell of an essential metabolite, in this case tryptophan, turns off its own manufacture and thus stops unneeded protein synthesis. As its name suggests, repressors are negative control mechanisms, shutting down operons

  • in the absence of a substrate (lactose in our example) or
  • the presence of an essential metabolite (tryptophan is our example).

However, some gene transcription in E. coli is under positive control.


UPDATE ON EcoCyc DATA

A team of curators performs curation of the E. coli literature on an ongoing basis. Based on regular PubMed searches, more than 1800 papers with some relevance to the biology of the E. coli laboratory strains are indexed in PubMed every year. Given limited resources, curation of this large amount of literature must be prioritized. At the same time, older entries in EcoCyc must be updated with more recent literature and new data types, such as Gene Ontology (GO) terms. We place the highest curation priority on publications that elucidate previously unknown gene functions and on publications that substantially advance our knowledge of E. coli biology. EcoCyc version 14.5 (September 2010) cites 20 284 publications, and users of the EcoCyc Web site can search the full text of 27 500 publications via Textpresso ( 1 ). Table 1 summarizes the current content of EcoCyc.

Overview of the current content of EcoCyc

Data Type . Number .
Genes 4489
Gene products covered by a mini-review 3666
Enzymes 1450
Metabolic reactions 1446
Compounds 2105
Transporters 252
Transport reactions 292
Transported substrates 207
Transcription factors 175
Transcription units 3409
With experimental evidence 1043
Regulatory interactions 5345
Transcription initiation 2746
Transcription attenuation 18
Enzyme modulation 2473
Other 108
Literature citations 20 284
Data Type . Number .
Genes 4489
Gene products covered by a mini-review 3666
Enzymes 1450
Metabolic reactions 1446
Compounds 2105
Transporters 252
Transport reactions 292
Transported substrates 207
Transcription factors 175
Transcription units 3409
With experimental evidence 1043
Regulatory interactions 5345
Transcription initiation 2746
Transcription attenuation 18
Enzyme modulation 2473
Other 108
Literature citations 20 284

Overview of the current content of EcoCyc

Data Type . Number .
Genes 4489
Gene products covered by a mini-review 3666
Enzymes 1450
Metabolic reactions 1446
Compounds 2105
Transporters 252
Transport reactions 292
Transported substrates 207
Transcription factors 175
Transcription units 3409
With experimental evidence 1043
Regulatory interactions 5345
Transcription initiation 2746
Transcription attenuation 18
Enzyme modulation 2473
Other 108
Literature citations 20 284
Data Type . Number .
Genes 4489
Gene products covered by a mini-review 3666
Enzymes 1450
Metabolic reactions 1446
Compounds 2105
Transporters 252
Transport reactions 292
Transported substrates 207
Transcription factors 175
Transcription units 3409
With experimental evidence 1043
Regulatory interactions 5345
Transcription initiation 2746
Transcription attenuation 18
Enzyme modulation 2473
Other 108
Literature citations 20 284

Because we prioritize new functional identifications and significant advances, the data from many publications are not immediately added to EcoCyc. However, our curation strategy includes systems-level updates on the functions of all gene products. These updates include literature searches for a set of genes (for example, all gene products involved in a metabolic pathway), and their curation will be updated with new and older literature and new data types, such as GO terms. Therefore, a significant number of older publications are later cited in EcoCyc. Abstracts and, if freely available, the full text of many E. coli publications, regardless of whether they have been cited in EcoCyc, are added to the Textpresso corpus and can be searched by EcoCyc users.

Update of transcriptional regulation data

EcoCyc knowledge of the transcriptional regulatory network is obtained from the literature, and is continually updated by the RegulonDB biocurator team ( 2 ). EcoCyc and RegulonDB releases are synchronized. In addition to our regular curation of knowledge from the literature, we have initiated the annotation of objects from high-throughput methodologies and computational predictions. Curation related to the regulation of transcription initiation is maintained with a delay of one or two months for each release.

Table 2 summarizes the information related to transcriptional regulation published in version 14.5, released in late September 2010. The number of objects has increased considerably since version 12.5 the growth is reported in column 3.

Summary of transcriptional regulation data in EcoCyc

Data type . Number . Number added since version 12.5 .
Transcription units 3409 53
Promoters 1878 124
Terminators 239 53
Transcription factors 175 12
Transcription factor binding sites 1940 310
Regulatory interactions 2697 355
Small molecule effectors 77 7
Data type . Number . Number added since version 12.5 .
Transcription units 3409 53
Promoters 1878 124
Terminators 239 53
Transcription factors 175 12
Transcription factor binding sites 1940 310
Regulatory interactions 2697 355
Small molecule effectors 77 7

Summary of transcriptional regulation data in EcoCyc

Data type . Number . Number added since version 12.5 .
Transcription units 3409 53
Promoters 1878 124
Terminators 239 53
Transcription factors 175 12
Transcription factor binding sites 1940 310
Regulatory interactions 2697 355
Small molecule effectors 77 7
Data type . Number . Number added since version 12.5 .
Transcription units 3409 53
Promoters 1878 124
Terminators 239 53
Transcription factors 175 12
Transcription factor binding sites 1940 310
Regulatory interactions 2697 355
Small molecule effectors 77 7

EcoCyc includes 175 transcription factors (TFs) with at least one experimentally characterized binding site or interaction, belonging to 33 evolutionary families of TFs. Curators author mini-review summaries for TFs and transcription units (TUs) that cover topics such as the regulatory mechanism, growth conditions in which regulation occurs, the cellular processes in which the regulated genes are involved, and a discussion of why those processes might be regulated together.

The 175 TFs are grouped as follows: Seven TFs are considered to be global regulators (ArcA, CRP, FIS, FNR, HNS, IHF, Lrp) 21 response regulators belong to two-component systems 42 TFs are included in the transport and catabolism of carbohydrates 17 TFs are related to processes such as transport, biosynthesis and catabolism of the amino acids 13 TFs are involved in transport and metabolism of different nitrogen sources and eight TFs are classified as metallo-regulators. Note that individual TFs can be involved in more than one function. The rest of the TFs are considered to be local regulators that control the transcription of genes involved in different cellular processes and functional classes.

Size correction of TF-binding sites (TFBSs)

Most TFs bind to small sequence motifs (7–25 nt) with different symmetries (inverted repeats, direct repeats or asymmetrical sequences) with a variable-size spacer sequence between them. The footprints of a few TFs are longer, from 35 to 60 bp. In general, these long regions do not contain conserved binding sites. Therefore, the binding motif has not been well characterized, even if these interactions are supported by strong evidence, such as: footprinting assay, electrophoretic mobility shift assay or mutations in the potential binding site. We have focused on these TFBSs, and have corrected and relocated several of them. We curated and relocated the binding sites for CytR, OxyR, FhlA based on computational tools [RSAT ( 3 )] that support the identification of overrepresented motifs in regulatory regions. The identification of consensus sequences is based on alignments of these upstream regions, performed by a curator, and on evidence obtained from the literature, including the similarity to the consensus sequence, data from footprinting assays, electrophoretic mobility shift assay, mutational analysis, computational analysis of these sequences and profiling of dependent gene expression.

For example, CytR binding sites were considered 60-bp long based on footprinting experiments. However, now the CytR-binding sites are described as octamer repeats (GTTGCATT) in direct or inverted orientation and preferably separated by 2 bp ( 4 , 5 ). With this information and based on computational analyses, we showed that the binding sites for CytR are overrepresented in the upstream region of the regulated genes. In a similar way, we have relocated, reassigned and corrected binding sites based on shorter binding motifs, for the Ada, YiaJ, NhaR and CaiF TFs.

GO terms

EcoCyc now contains assignments of GO ( 6 , 7 ) terms to a large number of E. coli genes. GO terms are assigned manually by EcoCyc curators during literature curation, and are imported twice a year from UniProt (which contains both computationally predicted and manually curated GO terms) and EcoliWiki. The total number of GO term annotations in EcoCyc is 43 288. Figure 1 shows the growth over time of experimental GO term annotations within EcoCyc. These data provide a lower bound on the number of E. coli genes with experimentally determined functions—a lower bound because GO term curation has been ongoing for a limited time and many well-characterized genes have not had their GO terms manually curated. Note also that many of the GO annotations with experimental evidence reflect approximate functional assignments.

Growth of experimental GO term annotations in EcoCyc over time. Data is grouped by database version, beginning with release 11.5 (August 2007) and ending with release 14.5 (September 2010). The first three bars in each group report the number of assignments of a GO term from each of the three primary GO categories with an EXP_IDA (inferred by direct assay) evidence code. In contrast, the last two bars report the number of genes that have at least one GO term annotation with an EXP or an EXP-IDA evidence code. EXP includes the EXP-IDA evidence codes as well as others such as EXP-IMP (inferred by mutant phenotype).

Growth of experimental GO term annotations in EcoCyc over time. Data is grouped by database version, beginning with release 11.5 (August 2007) and ending with release 14.5 (September 2010). The first three bars in each group report the number of assignments of a GO term from each of the three primary GO categories with an EXP_IDA (inferred by direct assay) evidence code. In contrast, the last two bars report the number of genes that have at least one GO term annotation with an EXP or an EXP-IDA evidence code. EXP includes the EXP-IDA evidence codes as well as others such as EXP-IMP (inferred by mutant phenotype).

Among the uses of GO terms in EcoCyc are retrieval of groups of genes that share a common biological process or molecular function (example: quick search for ‘cell division’, then click GO:0051301—cell division, for a list of all EcoCyc genes annotated to this term). GO terms can also be used in enrichment analysis of gene expression experiments, described below.

Protein features

Protein features are also both manually entered by EcoCyc curators, and are imported from UniProt before every EcoCyc release. UniProt features are derived from both experimental data and computational predictions. EcoCyc v14.5 contains 20 108 features, including enzyme active sites, phosphorylation sites and metal-ion binding sites. When protein features are available for a given protein, those features are displayed toward the bottom of the gene/protein page as annotations on the sequence (e.g. HypB). When importing both GO terms and protein features from UniProt, computationally predicted data are ignored when the same term or feature is present in EcoCyc with experimental evidence.

Protonation and reaction balancing

A barrier to the generation of flux-balance models from EcoCyc has been the fact that EcoCyc metabolites were inconsistently protonated, resulting in reactions that were not balanced for protons (although reactions have been balanced for all other elements for many years). Unbalanced reactions can result in the creation or disappearance of mass in flux-balance models, interfering with the proper functioning of such models. Therefore, we now computationally reprotonate all metabolites in EcoCyc [and MetaCyc ( 8 )] using the Marvin program (ChemAxon) before every release. We also computationally balance every reaction for protons by adding protons to the appropriate side of the reaction.


Conclusions

To evaluate the role of the global regulator Cra in the central carbon metabolism of E. coli K and E. coli B, the cra gene was deleted and the mutant strains were compared to the parental strains in their gene expression, growth and metabolic behaviour. The transcriptional changes caused by the deletion of cra gene support the assumption that Cra is a major regulatory molecule that has an effect on the expression of ppsA, aceBAK and acs in E.coli B (BL21), but these transcriptional changes did not affect the activity of the central carbon metabolism, suggesting that Cra does not act alone, rather it interacts with other pleiotropic regulators to create a network of metabolic effects. An unexpected outcome of this work is the finding that cra deletion in E. coli K-12 (JM109) caused transcription inhibition of the bet operon which is responsible for maintaining the salt concentration in the cells. In comparison, the bet operon expression was not affected in E. coli B (BL21). This property, together with this strain insensitivity to high glucose concentrations, makes this strain more resistant to environmental changes and may be one of the reasons for the growth properties of this strain.


Regulation of Protein Synthesis

The single chromosome of the common intestinal bacterium E.coli is circular and contains some 4.7 million base pairs.

The chromosome replicates in a bi-directional method, producing a figure resembling the Greek letter theta.

The promoter is the part of the DNA to which the RNA polymerase binds before opening the segment of the DNA to be transcribed.

Below is a gene map showing positions of some of the operons, such as trp and lac, on a bacterial chromosome:

A segment of the DNA that codes for a specific polypeptide is known as a structural gene.

These genes often occur together on a bacterial chromosome.

The close location of these genes allows for quick, efficient transcription of the mRNA's.

Often leader and trailer sequences, which are not translated, occur at the beginning and end of the region. E.coli can synthesize 1700 enzymes.

This small bacterium has the genes for 1700 different mRNAs. One of these genes is responsible for the formation of a sugar, lactose.

Lactose (a disaccharide found in milk) is digested by the enzyme ß-galactosidase. This enzyme ca be turned on and off and this was observed by noticing that it occurs in large quantities only when lactose, the substrate on which it operates, is present.

The Operon model

In the late 1950's, Fancois Jacob and Jacques Monod proposed the operon model of prokaryotic gene regulation. This work, which earnt them the Nobel prize, was based on the induction of ß galactosidase in E.coli.

Groups of genes coding for related proteins are arranged in units known as operons.

An operon consists of several sectors, namely:

  • An operator (o) region
  • A promoter (P) region
  • A regulator (i) gene
  • Structural genes (a,b,c. z)

The regulator gene codes for a repressor protein that binds to the operator, obstructing the promoter (suppressing transcription) of the structural genes.

The regulator does not have to be next to other genes in the operon. If the repressor protein is removed, transcription may occur.

This is an example using the lac Operon, found in E.coli and used in the metabolism of lactase for respiration.

The structural gene is responsible for the transcription process, forming mRNA, which is then used to synthesis the enzyme ß galactosidase.

The ribosomes are targeted to the starting point on the structural gene of the DNA.

mRNA is produced by transcription, and passes to the protein assembly sites in the bacterial cytoplasm.

Here, the mRNA is read by the ribosomes and a variety of polypeptide chains are constructed.

The structure and operation of an operon

Operons can either be inducible (promoters) or repressible according to the control mechanism.

In the 250 structural genes of E.coli, seventy-five different operons have been identified.

When a repressor molecule is present, this blocks the process of RNA polymerase, so preventing any transcription at this site.

However, when lactose, for example, is present in the food supplied to the bacterium, the repressor is removed and full transcription of the mRNA for lactose digestion (ß galactosidase) is possible.

Gene regulation in Eukaryotic organisms

Precisely how gene regulation occurs in eukaryotic cells is still unclear.

We do know that in transcription, proteins regulate the process by binding to specific sites on the DNA molecule.

However, the way in which regulation is achieved seems far more complex than in prokaryotes. One piece of evidence suggest that the influence of the regulator genes may be thousands of base pairs away from the promoter gene, unlike the close sequences found in E.coli.

Eukaryotes have a number of gene control mechanisms not found in prokaryotes. One of these is called DNA methylation.

In this example, it seems that some of the cytosine bases in the DNA are associated with a methyl (-CH3) group. It seems that where the genes are not expressed, the highest percentage of methylated genes are found but when drugs that inhibit methylation are applied, the genes start transcription.


Regulation of Cra protein level in E coli - Biology

Regulation and Control of Metabolism in Bacteria (page 1)

Bacterial Adaptation to the Nutritional and Physical Environment

Unlike plant and animal cells, most bacteria are exposed to a constantly changing physical and chemical environment. Within limits, bacteria can react to changes in their environment through changes in patterns of structural proteins, transport proteins, toxins, enzymes, etc., which adapt them to a particular ecological situation. For example, E. coli does not produce fimbriae for colonization purposes when living in a planktonic (free-floating or swimming) environment. Vibrio cholerae does not produce the cholera toxin that causes diarrhea unless it is in the human intestinal tract. Bacillus subtilis does not make the enzymes for tryptophan biosynthesis if it can find preexisting tryptophan in its environment. If E. coli is fed glucose and lactose together, it will use the glucose first because it takes two less enzymes to use glucose than it does to use lactose. The bacterium Neisseria gonorrhoeae will develop a sophisticated iron gathering and transport system if it senses that iron is in short supply in its environment.

Bacteria have developed sophisticated mechanisms for the regulation of both catabolic and anabolic pathways. Generally, bacteria do not synthesize degradative (catabolic) enzymes unless the substrates for these enzymes are present in their environment. For example, synthesis of enzymes that degrade lactose would be wasteful unless the substrate for these enzymes (lactose) is available in the environment. Similarly, bacteria have developed diverse mechanisms for the control of biosynthetic (anabolic) pathways. Bacterial cells shut down biosynthetic pathways when the end product of the pathway is not needed or is readily obtained by uptake from the environment. For example, if a bacterium could find a preformed amino acid like tryptophan in its environment, it would make sense to shut down its own pathway of tryptophan biosynthesis, and thereby conserve energy. However, in real bacterial life, the control mechanisms for all these metabolic pathways must be reversible, since the environment can change quickly and drastically.

Some of the common mechanisms by which bacterial cells regulate and control their metabolic activities are discussed in this chapter It is important for the reader to realize that most of these mechanisms have been observed or described in the bacterium, Escherichia coli, and they are mostly untested and unproved to exist in many other bacteria or procaryotes (although, whenever they are looked for, they are often found). The perceptive reader will appreciate that the origins of the modern science of molecular biology are found in the experiments that explained these regulatory processes in E. coli.

Conditions Affecting Enzyme Formation in Bacteria

As stated above, bacterial cells can change patterns of enzymes, in order to adapt them to their specific environment. Often the concentration of an enzyme in a bacterial cell depends on the presence of the substrate for the enzyme. Constitutive enzymes are always produced by cells independently of the composition of the medium in which the cells are grown. The enzymes that operate during glycolysis and the TCA cycle are generally constitutive: they are present at more or less the same concentration in cells at all times. Inducible enzymes are produced ("turned on") in cells in response to a particular substrate they are produced only when needed. In the process of induction, the substrate, or a compound structurally similar to the substrate, evokes formation of the enzyme and is sometimes called an inducer. A repressible enzyme is one whose synthesis is downregulated or "turned off" by the presence of (for example) the end product of a pathway that the enzyme normally participates in. In this case, the end product is called a corepressor of the enzyme.

Regulation of Enzyme Reactions

Not all enzymatic reactions occur in a cell to the same extent. Some substances are needed in large amounts and the reactions involved in their synthesis must therefore occur in large amounts. Other substances are needed in small amounts and the corresponding reactions involved in their synthesis need only occur in small amounts.

In bacterial cells, enzymatic reactions may be regulated by two unrelated modes: (1) control or regulation of enzyme activity (feedback inhibition or end product inhibition ), which mainly operates to regulate biosynthetic pathways and (2) control or regulation of enzyme synthesis, including end-product repression, which functions in the regulation of biosynthetic pathways, and enzyme induction and catabolite repression, which regulate mainly degradative pathways. The process of feedback inhibition regulates the activity of preexisting enzymes in the cells. The processes of end-product repression, enzyme induction and catabolite repression are involved in the control of synthesis of enzymes. The processes which regulate the synthesis of enzymes may be either a form of positive control or negative control. End-product repression and enzyme induction are mechanisms of negative control because they lead to a decrease in the rate of transcription of proteins. Catabolite repression is considered a form of positive control because it affects an increase in rates of transcription of proteins.

Table 1. Points for regulation of various metabolic processes. Bacteria exert control over their metabolism at every possible stage starting at the level of the gene that encodes for a protein and ending with alteration or modifications in the protein after it is produced. For example, variation in gene structure can vary the activity or production of a protein, just as modifications of a protein after it is produced can alter or change its activity. One of the most important sites for control of metabolism at the genetic level is regulation of transcription. At this level, in positive control mechanisms (e.g. catabolite repression), a regulatory protein has an effect to increase the rate of transcription of a gene, while in negative control mechanisms (e.g. enzyme induction or end product repression), a regulatory protein has the effect to decrease the rate of transcription of a gene. Sometimes this nomenclature may seem counter-intuitive, but molecular biologists have stuck us with it.

Although there are examples of regulatory processes that occur at all stages in molecular biology of bacterial cells (see Table 1 above), the most common points of regulation are at the level of transcription (e.g. enzyme induction and enzyme repression) and changing the activity of preexisting proteins. In turn, these levels of control are usually modulated by proteins with the property of allostery.

An allosteric protein is one which has an active (catalytic) site and an allosteric (effector) site. In an allosteric enzyme, the active site binds to the substrate of the enzyme and converts it to a product. The allosteric site is occupied by some small molecule which is not a substrate. However, when the allosteric site is occupied by the effector molecule, the configuration of the active site is changed so that it is now unable to recognize and bind to its substrate (Figure 1). If the protein is an enzyme, when the allosteric site is occupied, the enzyme is inactive, i.e., the effector molecule decreases the activity of the enzyme. There is an alternative situation, however. The effector molecule of certain allosteric enzymes binds to its allosteric site and consequently transforms the enzyme from an inactive to an active state (Figure 2). Some multicomponent allosteric enzymes have several sites occupied by various effector molecules that modulate enzyme activity over a range of conditions.

Figure 1. Example of an allosteric enzyme with a negative effector site. When the effector molecule binds to the allosteric site, substrate binding and catalytic activity of the enzyme are inactivated. When the effector is detached from the allosteric site the enzyme is active.


Figure 2. Example of an allosteric enzyme with a positive effector site. The effector molecule binds to the allosteric site resulting in alteration of the active site that stimulates substrate binding and catalytic activity.

Some allosteric proteins are not enzymes, but nonetheless have an active site and an allosteric site. The regulatory proteins that control metabolic pathways involving end product repression, enzyme induction and catabolite repression are allosteric proteins. In their case, the active site is a DNA binding site, which, when active, binds to a specific sequence of DNA, and which, when inactive, does not bind to DNA. The allosteric or effector molecule is a small molecule which can occupy the allosteric site and affect the active site. In the case of enzyme repression, a positive effector molecule (called a corepressor) binds to the allosteric regulatory protein and activates its ability to bind to DNA. In the case of enzyme induction a negative effector molecule (called an inducer) binds to the allosteric site, causing the active site to change conformation thereby detaching the protein from its DNA binding site.


16.2 Prokaryotic Gene Regulation

By the end of this section, you will be able to do the following:

  • Describe the steps involved in prokaryotic gene regulation
  • Explain the roles of activators, inducers, and repressors in gene regulation

The DNA of prokaryotes is organized into a circular chromosome, supercoiled within the nucleoid region of the cell cytoplasm. Proteins that are needed for a specific function, or that are involved in the same biochemical pathway, are encoded together in blocks called operons . For example, all of the genes needed to use lactose as an energy source are coded next to each other in the lactose (or lac) operon, and transcribed into a single mRNA.

In prokaryotic cells, there are three types of regulatory molecules that can affect the expression of operons: repressors, activators, and inducers. Repressors and activators are proteins produced in the cell. Both repressors and activators regulate gene expression by binding to specific DNA sites adjacent to the genes they control. In general, activators bind to the promoter site, while repressors bind to operator regions. Repressors prevent transcription of a gene in response to an external stimulus, whereas activators increase the transcription of a gene in response to an external stimulus. Inducers are small molecules that may be produced by the cell or that are in the cell’s environment. Inducers either activate or repress transcription depending on the needs of the cell and the availability of substrate.

The trp Operon: A Repressible Operon

Bacteria such as Escherichia coli need amino acids to survive, and are able to synthesize many of them. Tryptophan is one such amino acid that E. coli can either ingest from the environment or synthesize using enzymes that are encoded by five genes. These five genes are next to each other in what is called the tryptophan (trp) operon (Figure 16.3). The genes are transcribed into a single mRNA, which is then translated to produce all five enzymes. If tryptophan is present in the environment, then E. coli does not need to synthesize it and the trp operon is switched off. However, when tryptophan availability is low, the switch controlling the operon is turned on, the mRNA is transcribed, the enzyme proteins are translated, and tryptophan is synthesized.

The trp operon includes three important regions: the coding region, the trp operator and the trp promoter. The coding region includes the genes for the five tryptophan biosynthesis enzymes. Just before the coding region is the transcriptional start site . The promoter sequence, to which RNA polymerase binds to initiate transcription, is before or “upstream” of the transcriptional start site. Between the promoter and the transcriptional start site is the operator region.

The trp operator contains the DNA code to which the trp repressor protein can bind. However, the repressor alone cannot bind to the operator. When tryptophan is present in the cell, two tryptophan molecules bind to the trp repressor, which changes the shape of the repressor protein to a form that can bind to the trp operator. Binding of the tryptophan–repressor complex at the operator physically prevents the RNA polymerase from binding to the promoter and transcribing the downstream genes.

When tryptophan is not present in the cell, the repressor by itself does not bind to the operator, the polymerase can transcribe the enzyme genes, and tryptophan is synthesized. Because the repressor protein actively binds to the operator to keep the genes turned off, the trp operon is said to be negatively regulated and the proteins that bind to the operator to silence trp expression are negative regulators .

Link to Learning

Watch this video to learn more about the trp operon.

Catabolite Activator Protein (CAP): A Transcriptional Activator

Just as the trp operon is negatively regulated by tryptophan molecules, there are proteins that bind to the promoter sequences that act as positive regulators to turn genes on and activate them. For example, when glucose is scarce, E. coli bacteria can turn to other sugar sources for fuel. To do this, new genes to process these alternate sugars must be transcribed. When glucose levels drop, cyclic AMP (cAMP) begins to accumulate in the cell. The cAMP molecule is a signaling molecule that is involved in glucose and energy metabolism in E. coli. Accumulating cAMP binds to the positive regulator catabolite activator protein (CAP) , a protein that binds to the promoters of operons which control the processing of alternative sugars. When cAMP binds to CAP, the complex then binds to the promoter region of the genes that are needed to use the alternate sugar sources (Figure 16.4). In these operons, a CAP-binding site is located upstream of the RNA-polymerase-binding site in the promoter. CAP binding stabilizes the binding of RNA polymerase to the promoter region and increases transcription of the associated protein-coding genes.

The lac Operon: An Inducible Operon

The third type of gene regulation in prokaryotic cells occurs through inducible operons, which have proteins that bind to activate or repress transcription depending on the local environment and the needs of the cell. The lac operon is a typical inducible operon. As mentioned previously, E. coli is able to use other sugars as energy sources when glucose concentrations are low. One such sugar source is lactose. The lac operon encodes the genes necessary to acquire and process the lactose from the local environment. The Z gene of the lac operon encodes beta-galactosidase, which breaks lactose down to glucose and galactose.

However, for the lac operon to be activated, two conditions must be met. First, the level of glucose must be very low or non-existent. Second, lactose must be present. Only when glucose is absent and lactose is present will the lac operon be transcribed (Figure 16.5). In the absence of glucose, the binding of the CAP protein makes transcription of the lac operon more effective. When lactose is present, its metabolite, allolactose, binds to the lac repressor and changes its shape so that it cannot bind to the lac operator to prevent transcription. This combination of conditions makes sense for the cell, because it would be energetically wasteful to synthesize the enzymes to process lactose if glucose was plentiful or lactose was not available. It should be mentioned that the lac operon is transcribed at a very low rate even when glucose is present and lactose absent.

Visual Connection

In E. coli, the trp operon is on by default, while the lac operon is off. Why do you think this is the case?

trp receptor is repressed. Lactose, a sugar found in milk, is not always available. It makes no sense to make the enzymes necessary to digest an energy source that is not available, so the lac operon is only turned on when lactose is present.

If glucose is present, then CAP fails to bind to the promoter sequence to activate transcription. If lactose is absent, then the repressor binds to the operator to prevent transcription. If either of these conditions is met, then transcription remains off. Only when glucose is absent and lactose is present is the lac operon transcribed (Table 16.2).

Link to Learning

Watch an animated tutorial about the workings of lac operon here.


Abstract

Recent metabolic engineering practice was briefly reviewed including the case where a mixture of multiple sugars obtained from lignocellulose, etc. was used as a carbon source to produce a variety of biofuels and biochemicals for the realization of green society. In the wild type Escherichia coli, sequential utilization of carbon sources is observed as known as diauxie phenomenon due to carbon catabolite repression (CCR), where much attention has been focused on co-consumption of multiple sugars to improve the productivities of the target metabolites. Although co-consumption of multiple sugars can be attained by modulating phosphotransferase system (PTS) and mgsA, pgi, etc. in E. coli, the glucose uptake rate inherently became lower, and thus the productivity of such metabolite as ethanol may not be improved, where this may be improved by amplifying the non-PTS pathway genes such as galP and glk. It should be noted that the modulation of PTS gene might change the robustness from the systems biology point of view.


Cell lysis and protein purification

The next step following protein expression is often to isolate and purify the protein of interest. Protein yield and activity can be maximized by selecting the right lysis reagents and appropriate purification resin. We offer cell lysis formulations that have been optimized for specific host systems, including cultured mammalian, yeast, baculovirus-infected insect, and bacterial cells. Most recombinant proteins are expressed as fusion proteins with short affinity tags, such as polyhistidine or glutathione S-transferase, which allow for selective purification of the protein of interest. Recombinant His-tagged proteins are purified using immobilized metal affinity chromatography (IMAC) resins, and GST-tagged proteins are purified using a reduced glutathione resin.


Watch the video: Νεφρά: βλάπτει η ζωική πρωτεΐνη; (May 2022).


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