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15: Positive and negative control of gene expression - Biology

15: Positive and negative control of gene expression - Biology


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Operons

An operon is a cluster of coordinately regulated genes. It includes structural genes (generally encoding enzymes), regulatory genes (encoding, e.g. activators or repressors) and regulatory sites (such as promoters and operators). The type of control is defined by the response of the operon when no regulatory protein is present. In the case of negative control, the genes in the operon are expressed unless they are switched off by a repressor protein. Thus the operon will be turned on constitutively (the genes will be expressed) when the repressor in inactivated. In the case of positive control, the genes are expressed only when an active regulator protein, e.g. an activator, is present. Thus the operon will be turned off when the positive regulatory protein is absent or inactivated.

Table 4.1.1. Positive vs. negative control

Catabolic versus Biosynthetic Operons

Catabolic pathways catalyze the breakdown of nutrients (the substrate for the pathway) to generate energy, or more precisely ATP, the energy currency of the cell. In the absence of the substrate,there is no reason for the catabolic enzymes to be present, and the operon encoding them is repressed. In the presence of the substrate, when the enzymes are needed, the operon is induced or de-repressed.

Table 4.1.2. Comparison of catabolic and biosynthetic operons
Operon encodesAbsence ofEffectPresence ofEffect
catabolic enzymessubstraterepressedsubstratederepressed (induced)
biosynthetic enzymesproductinducedproductrepressed

For example, the lac operon encodes the enzymes needed for the uptake (lactose permease) and initial breakdown of lactose (the disaccharide b-D-galactosyl-1->4-D-glucose) into galactose and glucose (catalyzed by b-galactosidase). These monosaccharides are broken down to lactate (principally via glycolysis, producing ATP), and from lactate to CO2 (via the citric acid cycle), producing NADH, which feeds into the electron-transport chain to produce more ATP (oxidative phosphorylation). This can provide the energy for the bacterial cell to live. However, the initial enzymes (lactose permease and b-galactosidase) are only needed, and only expressed, in the presence of lactose and in the absence of glucose. In the presence of the substrate lactose, the operon in turned on, and in its absence, the operon is turned off.

Anabolic, or biosynthetic, pathways use energy in the form of ATP and reducing equivalents in the form of NAD(P)H to catalyze the synthesis of cellular components (the product) from simpler materials, e.g. synthesis of amino acids from small dicarboxylic acids (components of the the citric acid cycle). If the cell has plenty of the product already (in the presence of the product), the the enzymes catalyzing its synthesis are not needed, and the operon encoding them is repressed. In the absence of the product, when the cell needs to make more, the biosynthetic operon is induced. E.g., the trpoperon encodes the enzymes that catalyze the conversion of chorismic acid to tryptophan. When the cellular concentration of Trp (or Trp-tRNAtrp) is high, the operon is not expressed, but when the levels are low, the operon is expressed.

Inducible versus repressible Operons

Inducible operons are turned on in reponse to a metabolite (a small molecule undergoing metabolism) that regulates the operon. E.g. the lac operon is induced in the presence of lactose (through the action of a metabolic by-product allolactose). Repressible operons are switched off in reponse to a small regulatory molecule. E.g., the trpoperon is repressed in the presence of tryptophan. Note that in this usage, the terms are defined by the reponse to a small molecule. Although lac is an inducible operon, we will see conditions under which it is repressed or induced (via derepression).

Table 4.1.3.

Map of the E. colilac operon

Figure 4.1.1.

  1. Promoters = p= binding sites for RNA polymerase from which it initiates transcription. There are separate promoters for the lacIgene and the lacZYAgenes.
  2. Operator = o = binding site for repressor; overlaps with the promoter for lacZYA.
  3. Repressor encoded by lacIgene
  4. Structural genes: lacZYA

lacZ encodes b-galactosidase, which cleaves the disccharide lactose into galactose and glucose.

lacYencodes the lactose permease, a membrane protein that faciltitates uptake of lactose.

lacAencodes b-galactoside transacetylase; the function of this enzymes in catabolism of lactose is not understood (at least by me)

C. Negative control

The lac operon is under both negative and positive control. The mechanisms for these will be considered separately.

1. In negative control, the lacZYAgenes are switched off by repressor when the inducer is absent (signalling an absence of lactose). When the repressor tetramer is bound to o, lacZYAis not transcribed and hence not expressed.

Figure 4.1.2. Repressed lac operon

2. When inducer is present (signalling the presence of lactose), it binds the repressor protein, thereby altering its conformation, decreasing its affinity for o, the operator. The dissociation of the repressor-inducer complex allows lacZYAto be transcribed and therefore expressed.

Figure 4.1.3. Induction of the lac operon by derepression.

Inducers

The natural inducer (or antirepressor), is allolactose, an analog of lactose. It is made as a metabolic by-product of the reaction catalyzed by b-galactosidase. Usually this enzyme catalyzes the cleavage of lactose to galactose + glucose, but occasionally it will catalyze an isomerization to form allolactose, in which the galacose is linked to C6 of glucose instead of C4.

A gratuitous inducer will induce the operon but not be metabolized by the encoded enzymes; hence the induction is maintained for a longer time. One of the most common ones used in the laboratory is a synthetic analog of lactose called isopropylthiogalactoside (IPTG). In this compound the b-galactosidic linkage is to a thiol, which is not an efficient substrate for b-galactosidase.

E. Regulatory mutants

Regulatory mutations affect the amount of all the enzymes encoded by an operon, whereas mutations in a structural gene affects only the activity of the encoded (single) polypeptide.

Repressor mutants

  • a. Wild-type strains (lacI+) are inducible.
  • b. Most strains with a defective repressor (lacI-) are constitutive, i.e. they make the enzymes encoded by the lac operon even in the absence of the inducer.
  • c. Strains with repressor that is not able to interact with the inducer (lacIS) are noninducible. Since the inducer cannot bind, the repressor stays on the operator and prevents expression of the operon even in the presence of inducer.
  • d. Deductions based on phenotypes of mutants
Table 4.1.4. Phenotypes of repressor mutants
b-galactosidasetransacetylase
Genotype-IPTG+IPTG-IPTG+IPTGConclusion
I+Z+A+<0.1>100<1>100Inducible
I+Z-A+<0.1><0.1><1>100lacZencodes b-galactosidase
I-Z+A+1001009090Constitutive
I+Z-A+ /F' I-Z+A+<0.1>100<1>200I+ >I- in trans
IsZ+A+<0.1><1><1><1>Noninducible
IsZ+A+ /F' I+Z+A+<0.1>1<1>1Is>.I+ in trans
  1. The wild-type operon is inducible by IPTG.
  2. A mutation in lacZaffects only b-galactosidase, not the transacetylase (or other products of the operon), showing that lacZis a structural gene.
  3. A mutation in lacIaffects both enzymes, hence lacIis a regulatory gene. Both are expressed in the absence of the inducer, hence the operon is constitutively expressed (the strain shows a constitutive phenotype).
  4. In a merodiploid strain, in which one copy of the lac operon is on the chromosome and another copy is on an F' factor, one can test for dominance of one allele over another. The wild-type lacI+is dominant over lacI-intrans. In a situation where the only functional lacZgene is on the same chromosome as lacI-, the functional lacI still causes repression in the absence of inducer.
  5. The lacISallele is noninducible.
  6. In a merodiploid, the lacISallele is dominant over wild-type in trans.

e. The fact that the product of the lacIgene is trans-acting means that it is a diffusible molecule that can be encoded on one chromosome but act on another, such as the F' chromosome in example (d) above. In fact the product of the lacIgene is a repressor protein.

2. Operator mutants

a. Defects in the operator lead to constitutive expression of the operon, hence one can isolate operator constitutive mutations, abbreviated oc. The wild-type o+is inducible.

b. Mutations in the operator are cis-acting; they only affect the expression of structural genes on the same chromosome.

(1)The merodiploid I+ocZ+/I+o+Z- [this is an abbreviation for lacI+oclacZ+/lacI+o+lacZ-] expresses b-galactosidase constitutively. Thus oc is dominant to o+ when oc is in cisto lacZ+.

(2)The merodiploid I+ocZ-/I+o+Z+ is inducible for b-galactosidase expression. Thus o+ is dominant to oc when o+ is in cisto lacZ+.

(3)The allele of othat is in cisto the active reporter gene (i.e., on the same chromosome as lacZ+ in this case) is the one whose phenotype is seen. Thus the operator is cis-acting, and this property is referred to as cis-dominance. As in most cases of cis-regulatory sequences, these are sites on DNA that are required for regulation. In this case the operator is a binding site for the trans-acting repressor protein.

Interactions between Operator and Repressor

Sequence of operator

The operator overlaps the start the site of transcription and the promoter. It has a dyad symmetry centered at +11. Such a dyad symmetry is commonly found within binding sites for symmetrical proteins (the repressor is a homotetramer). The sequence of DNA that consititutes the operator was defined by the position of oC mutations, as well as the nucleotides protected from reaction with, e.g. DMS, upon binding of the repressor.

Figure 4.1.4.

2. Repressor

a. Purification

(1)Increase the amount of repressor in the starting material by over-expression.

A wild-type cell has only about 10 molecules of the repressor tetramer. Isolation and purification of the protein was greatly aided by use of mutant strain with up-promoter mutations for lacI, so that many more copies of the protein were present in each cell. This general strategy of over-producing the protein is widely used in purification schemes. Now the gene for the protein is cloned in an expression vector, so that the host (bacteria in this case) makes a large amount of the protein - often a substantial fraction of the total bacterial protein.

(2)Assays for repressor

[1]Binding of radiolabeled IPTG (gratuitous inducer) to repressor

[2]Binding of radiolabeled operator DNA sequence to repressor. This can be monitored by the ability of the protein-DNA complex to bind to nitrocellulose (whereas a radiolabeled mutant operator DNA fragement, oc, plus repressor will not bind). Electrophoretic mobility shift assays would be used now in many cases.

[3]This ability of particular sequences to bind with high affinity to the desired protein is frequently exploited to rapidly isolate the protein. The binding site can be synthesized as duplex oligonucleotides. These are ligated together to form multimers, which are then attached to a solid substrate in a column. The desired DNA-binding protein can then be isolated by affinity chromatography, using the binding site in DNA as the affinity ligand.

b. The isolated, functional repressor is a tetramer; each of the four monomers is the product of the lacI gene (i.e. it is a homotetramer).

c. The DNA-binding domainof the lac repressor folds into a helix-turn-helixdomain. We will examine this structural domain in more in Chapter III. It is one of the most common DNA-binding domains in prokaryotes, and a similar structural domain (the homeodomain) is found in some eukaryotic transcriptional regulators.

3. Contact points between repressor and operator

a. Investigation of the contact points between repressor and the operator utiblized the same techniques that we discussed previously for mapping the binding site of RNA polymerase on the promoter, e.g. electrophoretic mobility shift assays (does the DNA fragment bind?), DNase footprints (where does the protein bind?) and methylation interference assays (methylation of which purines will prevent binding?). Alternative schemes will allow one to identify sites at which methylation is either prevented or enhanced by the binding of the repressor. These techniques provide a biochemical defintion of the operator = binding site for repressor.

b. The key contact points (see Figure 4.1.4.):

(1)are within the dyad symmetry.

(2)coincide (in many cases) with nucleotides that when mutated lead to constitutive expression. Note that the latter is a genetic definition of the operator, and it coincides with the biochemically-defined operator.

(3)tend to be distributed symmetrically around the dyad axis (+11).

(4)are largely on one face of the DNA double helix.

c. The partial overlap between the operator and the promoter initially suggested a model of steric interference to explain the mechanism of repression. As long a repressor was bound to the operator, the polymerase could not bind to the promoter. But, as will be explored in the next chapter, this is notthe case. RNA polymerase canbind to the lacpromoter even when repressor is boudn to the lac operator. However, the polymerase cannot initiatetranscription when juxtaposed to the repressor.

4. Conformational shift in repressor when inducer binds

a. The repressor has two different domains, one that binds to DNA ("headpiece" containing the helix-turn-helix domain) and another that binds to the inducer (and other subunits) (called the "core). These are connected by a "hinge" region.

b. These structural domains can be distinguished by the phenotypes of mutations that occur in them.

lacI-dprevents binding to DNA, leads to constitutive expression.

lacISprevents binding of inducer, leads to a noninducible phenotype.

c. Binding of inducer to the "core" causes an allosteric shift in the repressor so that the "headpiece" is no longer able to form a high affinity complex with the DNA, and the repressor can dissociate (go to one of the many competing nonspecific sites).

Positive control: "catabolite repression"

1. Catabolite repression

a. Even bacteria can be picky about what they eat. Glucose is the preferred source of carbon for E. coli; the bacterium will consume the available glucose before utilizing alternative carbon sources, such as lactose or amino acids.

b. Glucose leads to repression of expression of lacand some other catabolic operons. This phenomenon is called catabolite repression.

2. Two components are needed for this form of regulation

a. cAMP

[1]In the presence of glucose, the [cAMP] inside the cell decreases from 10-4 M to 10-7 M. A high [cAMP] will relieve catabolite repression.

[2]cAMP synthesis is catalyzed by adenylate cyclase (product of the cyagene)

ATP ® cAMP + PPi

b. Catabolite Activator Protein = CAP

[1]Product of the capgene, also called crp(cAMP receptor protein).

[2]Is a dimer

[3]Binds cAMP, and then the cAMP-CAP complex binds to DNA at specific sites

3. Binding site for cAMP-CAP

a. In the lac operon, the binding site is a region of about 20 bp located just upstream from the promoter, from -52 to -72.

b. The pentamer TGTGA is an essential element in recognition. For the lac operon, the binding site is a dyad with that sequence in both sides of the dyad.

c. Contact points betwen cAMP-CAP and the DNA are close to or coincident with mutations that render the lacpromoter no longer responsive to cAMP-CAP.

d. cAMP-CAP binds on one face of the helix.

Figure 4.1.5. Binding site for cAMP-CAP

4. Binding of cAMP-CAP to its site will enhance efficiency of transcription initiation at promoter

a. The lacpromoter is not a particularly strong promoter. The sequence at -10, TATGTT, does not match the consensus (TATAAT) at two positions.

b. In the presence of cAMP-CAP, the RNA polymerase will initiate transcription more efficiently.

c. The lacUV5 promoter is an up-promoter mutation in which the -10 region matches the consensus. The lac operon driven by the UV5 promoter will achieve high level induction without cAMP-CAP, but the wild-type promoter requires cAMP-CAP for high level induction.

Figure 4.1.6. Regulatory region of lac operon, including CAP binding site

5. Mode of action of cAMP-CAP

a. Direct positive interaction with RNA polymerase. The C-terminus of the a subunit is required for RNA polymerase to be activated by cAMP-CAP. This will be explored in more detail in Chapter 16.

b. cAMP-CAP bends the DNA about 90o.

Figure 4.1.7. DNA (top helical structure) is bent by the CAP dimer .

Some generalities

  • Repressors, activators and polymerases interact primarily with one face of the DNA double helix.
  • Regulatory proteins, such as activators and repressors, are frequently symmetrical and bind symmetrical sequences in DNA.
  • RNA polymerases are not symmetrical, and the promoters to which they bind also are asymmetrical. This confers directionality on transcription.

Regulation of Gene Expression in Prokaryotes and Eukaryotes

Let us make an in-depth study of the gene expression regulation. After reading this article you will learn about 1. Regulation of Gene Expression in Prokaryotes and 2. Regulation of Gene Expression in Eukaryotes.

Gene is a part of DNA that specifies a protein/RNA. All the proteins/RNA are not required by the cell all the time. Some proteins are required at some time and yet other proteins are required at another time. Moreover these proteins are required in lesser quantities at one time, yet at other times they may be required in higher quantities. There are yet another class of proteins which are constantly (always) present in the cell, like the enzymes of the TCA cycle.

Therefore genes can be conveniently grouped under two classes:

1. Constitutive genes:

Those genes whose products are constantly present in the cell are called constitutive genes or housekeeping genes.

2. Inducible genes:

Those genes whose products vary with time and need, both in their presence and concentration are called inducible genes or those genes whose products (proteins/RNA) are in­duced by some inducer molecule. The activity of the constitutive genes is not regulated as their products don’t vary much with time, whereas the activity of inducible genes is always regulated. The regulation is primarily at the level of transcription. The gene or a set of related genes are switched on or off as per the need of the cell. These changes are brought about by some proteins or modulator.

If a particular protein/compound puts a gene into operation then that protein is called stimulatory protein/compound and the process is called positive regulation. If a protein/compound stops the operation of a gene then it is called the repressor protein/ compound and this process is referred to as negative regulation, ex. a steroid hormone acts as a positive modulator, wherein its presence enhances the rate of gene expression.

As soon as the hormone is destroyed the gene expression diminishes. The mechanism of regulation, though similar in the prokaryotes and eukaryotes, it differs in some aspects. Hence regulation of gene expression in prokaryotes and eukaryotes will be taken separately.

Regulation of Gene Expression in Prokaryotes:

Many prokaryotic genes are regulated in units called operons. Operon is unit of genetic expression consisting of one or more related genes and sequences (gene) controlling them, which includes the operator and promoter sequences that regulate their transcription.

It is the operon for utilization and metabolism of lactose in bacteria.

It consists of the following set of genes:

PI = The promoter gene for regulatory genes

I = The gene for regulatory protein (repressor protein)

P = The promoter sequence for the related genes

O = Operator sequence for these genes

Z = The first gene for utilization of lactose, which forms the enzyme beta-galactosidase

Y = The second gene for the membrane protein galactoside permease

A = The third gene for the enzyme thiogalactoside trans-acetylase

This complete set of sequences (i.e. the operon) helps in switching on/off, the machinery for the utilization of the carbohydrate-lactose by the bacteria E. coli. When glucose is present in the media where the cell is growing, then the lac operon is switched off and when the medium is devoid of glucose, and instead lactose is present as the sole source of carbon, then the Lac operon becomes operational.

The transcription by RNA polymerase begins at the promoter site i.e. the enzyme binds to the promoter and moves along the DNA towards the structural genes of the operon to transcribe the mRNA for these genes and in this process it passes through the operator region of the operon.

Under all circumstances i.e. whether glucose or lactose is to be utilized by the cell, the I gene of the lac operon synthesizes a protein called repressor protein. This protein binds to the operator site in the DNA and thus prevents the movement of the RNA polymerase beyond this point (site), which results in the inhibition of the synthesis of the structural genes Z, Y and A.

Thus, when the cell is utilizing glucose as the only carbon source, the lac operon is switched off. Then, if the cell shifts over to the utilization of lactose as the carbon source then lactose is first converted to allolactose by the enzyme beta-galactosidase (which is always present in the cell in a few copies, irrespec­tive of glucose or lactose is being utilized), and this allolactose acts as a positive modulator or inducer for the lac operon.

Here the allolactose binds to the repressor protein present at the operator site resulting in the release of the repressor protein from the operator site thereby permitting the enzyme RNA polymerase to pass freely through this operator site from the promoter site and thus transcribe all three structural genes Z, Y, & A.

The activity of the lac operon is not only dependent upon the binding and release of repressor molecule (with modulator) but it is also cAMP dependent. When glucose is low in the media/cell, then the cellular cAMP concentration increases. This increased amount of cAMP results in its binding at a particular site (sequences) on the promoter.

The promoter site can be divided into two parts:

(1) The site for the binding of RNA polymerase

(2) The site for a protein called catabolite gene activator protein (CAP).

The RNA polymerase can bind to the promoter site only if the CAP is bound to the promoter sequence and CAP can bind to the promoter only if cAMP is bound to it and cAMP binds to CAP only when its cellular concentration increases, which occurs when the cell is devoid of glucose and hence this facilitates the utilization of these sugars and the presence of lactose converts it to allolactose.

This acts as a positive modulator for switching on the lac operon genes by releasing the repressor protein from the operator site and producing the products of the three structural genes which produces:

(1) The membrane protein β-galactoside permease, that enhances the uptake of lactose by the cells

(2) β-galactosidase which hydrolysis lactose to allolactose and then to glucose and galactose

(3) The enzyme thiogalacatosidase-transacetylase, whose function is unknown.

When glucose is again available to the cell the cAMP concen­tration decreases in the cytosol, resulting in its release from the CAP, this in turn results in the release of CAP from promoter site, which in turn results in release of the enzyme RNA polymerase from the promoter site and further prevents its binding to promoter.

This again results in the diminished synthesis of the structural genes, one of which is beta-galactosidase, that results in low production of allolactose (or no synthesis of allolactose), this is turn results in the repressor protein (formed from I gene) being devoid of the modulator and thus is free to bind at the operator site thereby prevent the movement of RNA polymerase and thus resulting in the inhibition of lac operon.

Each and every metabolite has got its own operon, with different number of structural genes and when­ever the genes for that metabolite are required it is switched on by a similar mechanism as that of the lac operon and switched off whenever not required.

The other operons and their details are as under:

All of the operons found in the bacteria do not function only by completely switching on or off their genes. Some operons function at differential rates depending upon the need of the cell by a mechanism called the transcription attenuation i.e. slowing down of the rate of synthesis of enzymes, ex. those enzymes involved in the synthesis of amino acids (His).

Transcription attenuation is a process in which transcription is initiated normally but is abruptly halted before the complete operon genes are transcribed. The frequency with which transcription is attenuated depends upon the cellular concentration of that particular amino acid for which the operon is meant for.

Attenuation of His operon:

In bacteria, transcription and translation are closely coupled. The rate at which RNA is transcribed and the rate at which that protein is translated is almost the same. Most of the transcribed RNAs for amino acid metabolism in the cell contain various complementary intra base pairing sequences. For example the following is the part of RNA that is being transcribed for His operon, which is also simultaneously being translated.

The sequence 2 and 3 are complementary and can base pair with each other. Likewise sequences 3 and 4 are also complementary and can also base pair with each other. If 2 and 3 bases pair, then transcription can proceed normally and if 3 and 4 bases pair the transcription is terminated, just like the termination of transcription due to appearance of a hair pin structure in DNA. The base pairing between the sequences 2 & 3 or 3 & 4 is dependent upon the rate of translation of the mRNA, which in turn is dependent upon the concentration of His-tRNA His that reflects the concentration of histidine in the cell.

If the concentra­tion of His-tRNA His is more and the rate of translation is very fast such that it passes the 2 nd site before site 3 is transcribed, then this results in the site 3 base pairing with site 4 as soon as it is transcribed resulting in the termination of transcription.

On the other hand when the His-tRNA His concentration is low, the rate of translation is very slow and thus the process of translation does not pass the 2 nd site on mRNA by the time site (sequences) 3 rd is transcribed then this result in the continuation of transcription because this will result in the 2 & 3 sites base pairing and so site 3 is not free for base pairing with site 4. Thus this results in a continuous operation of His operon.

Regulation of Gene Expression in Eukaryotes:

The genes in eukaryotes are also regulated in more or less the same manner as that of prokaryotes, but the regulation is mostly positive and very rarely negative regulation is seen. In higher eukaryotes the regulation of gene expression is solely by positive modulation and negative inhibition of the genes/operon is totally absent.

However in yeast some genes are regulated by negative modulation. Further, there is a physical separation between the process of transcription and translation is eukaryotes as transcription takes place in the nucleus and translation occurs in the cytosol.

The gene regulation is only by positive regulation. Most of the genes are normally inactive in eukaryotes i.e. RNA polymerases cannot bind to the promoters. The cells synthesize only the selected group of activator proteins needed to activate transcription of the small subset of genes required in that cell.

There are at least five regulatory sites for RNA polymerase promoter sites in higher eukaryotes designated as (a) TATA box (b) GC box and (c) CAT box. In yeast there are two types of promoter sequences i.e. TATA box and UAS i.e. upstream activator sequence.

These sequences are the binding sites for the transcription factors called TF-II-D that is required for RNA polymerase binding. Each of these sequences are recognised and bound specifically by one or more regulatory proteins called transcription factors. These regulatory sequences are about 1000 bases away form the main gene, thus to activate the main gene a protein-protein interaction is required which can reach the main gene sequence.


Components of Lac-Operon

An operon primarily consists of two elements or genes:

Regulatory Elements

It includes the following regions:

  • Promotor Region: It codes the Lac-P gene. It lies between the regulator and the operator. RNA-polymerase binds to this site, as a promoter region initiates transcription. It is 100 base pairs long. It consists of palindromic sequences. This site promotes and controls the transcription of structural genes or m-RNA. The regulatory genes of the repressor regulate the functioning of the promoter region.
  • Operator Region: It codes the Lac-O gene. It lies between a promoter and the structural gene (Lac-Z). It contains an operator switch, which decides whether transcription should take place or not. The regulatory gene binds to the operator.
  • Regulator Region: It codes for regulator gene (Lac-I) that controls the activity of promotor and an operator gene. This regulatory gene produces regulatory proteins known as “Repressor proteins” that can bind to the promoter and operator.

Structural Elements

These are the regions of DNA, which contain genes for the protein synthesis, and they are of three kinds:

  • Lac-Z: Encodes for the enzyme beta-galactosidase.
    Function: Beta-galactosidase brings about the hydrolysis of lactose into galactose and glucose subunits.
  • Lac-Y: Encodes for the enzyme lactose permease.
    Function: Lactose permease brings lactose into the cell.
  • Lac-A: Encodes for the enzyme thiogalactoside transacetylase.
    Function: Thiogalactoside transacetylase function is not very clear, but it assists the activity of an enzyme beta-galactosidase.

These three, i.e. Lac Z, Y and A genes, are present adjacent to each other. Therefore, all the elements like promotor, operator, repressor and structural genes together form a unit called Operon.

Control of Gene Expression in Prokaryotes

In prokaryotes, the Lac-operon system is controlled by two ways:

Positive Control of Lac-Operon

It is also called Positive inducible system and includes the following steps:

  1. Firstly, a regulatory gene expresses the repressor protein.
  2. After that, repressor proteins are produced by the expression of a regulatory gene.
  3. A repressor protein has binding sites for the operator and the inducer (lactose).
  4. When lactose is present as an inducer, it binds with the repressor protein and forms R+I complex.
  5. After the binding of inducer to the repressor, the complex blocks the binding of the repressor to the operator.
  6. As the repressor protein does not block the operator, the RNA polymerase binds to the promotor and moves further to transcribe mRNA.

This concept is known as switch on of Lac-operon (by the presence of an inducer).

Negative Control of Lac-Operon

It is also called Negative control of repressor system. It includes the following steps:

  1. First, the regulatory gene is expressed by the repressor.
  2. A repressor protein is produced after the expression of a regulatory gene.
  3. In the absence of inducer or lactose, the repressor protein directly binds to an operator.
  4. This blocks the movement of RNA polymerase and its attachment to the promoter.
  5. At last, mRNA transcription will not occur.

This concept is known as switch off of Lac-operon (by the absence of an inducer).


Methods

Algal strains, growth conditions and cell treatment

The following Chlamydomonas reinhardtii strains were used: wild-type cw15–325 (mt+, cw15, arg7), which was kindly provided by Dr. M. Schroda (University of Kaiserslautern, Germany) and transformants with reduced THB1 obtained from cw15–325 amiTHB1–11 (mt+, cw15), amiTHB1–14 (mt+, cw15) and amiTHB1–23 (mt+, cw15) [29]. The 305 mutant (mt nit1) affected in NAD(P) H-NR activity and without diaphorase-NR activity was originally obtained from the wild type 6145c (mt ) [30]. The 305 and 6145 strains were kindly provided by Dr. E. Fernández (University of Cόrdoba, Spain).

Cells were grown mixotrophically in tris-acetate-phosphate (TAP) medium (https://www.chlamycollection.org/methods/media-recipes/tap-and-tris-minimal/) containing 7.5 mM NH4Cl instead of NH4NO3 under continuous illumination with white light (fluence rate of 45 μmol m − 2 s − 1 ) at 22 °C with constant orbital agitation at 90 rpm. The TAP medium was supplemented with 100 mg L − 1 of arginine when required. Cells were collected at the midexponential phase of growth by centrifugation (4000 g, 5 min), washed twice with 10 mM potassium phosphate, pH 7, before being transferred to the induction media containing the different sources of nitrogen and chemicals. At each harvesting times the number of viable cells were counted microscopically with use of 0.05% (v/v) Evans blue (DIA-M, Russia) as described [31]. Non-viable (stained) and viable (unstained) cells were counted. Four-hundred cells from each sample were scored for three biological replicates.

Determination and calculations of total chlorophyll (μg/ml) were performed as previously described [29, 32].

The compounds DEA-NONOate [2-(N, Ndiethylamino)-diazenolate 2-oxide sodium salt] and ODQ [1H-(1,2,4])oxadiazolo(4,3-a) quinoxalin-1-one] are from Sigma-Aldrich.

Gene expression analysis

The total RNA was isolated with Trizol according to the manufacturer’s instructions (Invitrogen, USA). To remove genomic DNA, the RNA samples were treated with RNase-Free DNase I (Fermentas). Subsequently, RNA concentration and purity (260/280 nm ratio) was determined using spectrophotometer (SmartSpec Plus, Bio-Rad).

Revert Aid HMinus First Strand cDNA Synthesis Kit (Thermo Scientific) was used for reverse transcription reaction. The primer pairs for RTqPCR are given in Additional file 1: Table S1. RT qPCR was performed with a CFX96 Real-Time PCR Detection System (Bio Rad) using SYBR Green I according to [33]. Gene expression ratios were calculated with the ΔΔCt method [34]. The RACK1 (receptor of activated protein kinase C Cre13.g599400) gene was chosen as the control housekeeping gene. All reactions were performed in triplicate with at least three biological replicates. Significant differences between experiments were evaluated statistically by standard deviation and Student’s t-test methods.

Protein gel blot analysis

The protein content was determined with amido black staining and protein gel blot analysis was performed as described [33, 35]. After separation by SDS-PAGE on a 12% polyacrylamide gel (w/v), the proteins were transferred to nitrocellulose membranes (Carl Roth, Karlsruhe) with use of semidry blotting (Trans-blot SD BioRad). The dilutions of the primary antibodies used were as follows: 1:5,000 anti-CrPII and 1:2000 anti-HSP70B. As a secondary antibody, the horseradish peroxidase-conjugated anti-rabbit serum (Sigma) was used at a dilution of 1:10,000. The peroxidase activity was detected via an enhanced chemiluminescence assay (Roche). For quantification, films were scanned using Bio-Rad ChemiDocTMMP Imaging System, and signals were quantified using the Image LabTM software (version 5.1).

Nitrate determination

After eliminating the cells by centrifugation at 3000 g, nitrate concentrations in the medium were determined by dual-wavelength ultraviolet spectrophotometry as A220 - 2A275 using standard curve [36]. For the measurements, media with 4 mM nitrate were diluted 50-fold. Values were obtained from at least three biological replicates each replicate was analyzed three times. Student’s t-tests were used for statistical comparisons. P-values of< 0.05 were considered as significant.

Measurement of NO

Cells were treated with DEA-NONOate or nitrite, then they were incubated with in the presence of 1 μM (4-amino-5-methylamino-2′7’-difluorofluorescein diacetate) dye (DAF-FM DA, Sigma-Aldrich), at concentration of 45 μg/ml chlorophyll. After 15 min the cells were washed, resuspended in indicated medium and used for the fluorometric detection of NO. The supernatant was collected in a test tube and then used to detect NO in the medium. The measurement of NO was carried out with a microplate reader CLARIOstar (BMG) as described [29]. The excitation and emission wavelengths for the NO indicator were 483 ± 14 and 530 ± 30 nm, respectively. Fluorescence intensity was calculated as arbitrary units per chlorophyll or protein as described previously [29].

NO detection by confocal microscopy

Cells were treated as described above. Images were acquired with a Leica TCS-SP5 confocal microscope (Leica-Microsystems, Germany) as described [29]. All experiments were performed in triplicate.


Regulation of Gene Expression in Prokaryotes (With Diagram)

(ii) Those that are synthesized only after a specific stimulation. The first type was named constitutively synthesized and the latter the inducible enzymes.

Analyzing a variety of E. coli that were defective for the induction of the lactose utilizing enzymes, Jacob and Monod hit upon the possible molecular mechanism that controls the repression and de-repression of a set of genes. The E. coli requires a set of three genes to be able to metabolize lactose. When a little lactose is added to a glucose-free growth medium, it is seen that these three lactose utilizing genes (lac genes) named lac z, lac y and lac a are synthesized simultaneously.

The product of lac z is the enzyme β-gaIactosidase that catalyzes the conversion of lactose into galactose and glucose. These genes are note expressed in the absence of lactose. Jacob and Monod (1961) proposed the operon model to explain the genetic basis of induction and repression of lac genes in prokaryotes. They were awarded Nobel Prize for this work in 1965.

The Operon:

1. Operons are segments of genetic material (DNA) that function as regulated unit that can be switched on or off.

2. An operon consists of minimum four types of genes: regulator, operator, promoter and structural (Fig. 8.4.A).

3. Regulator gene is a gene which forms a biochemical for suppressing the activity of operator gene.

4. Operator gene is a gene which receives the product of regulator gene. It allows the functioning of the operon when it is not covered by the biochemical produced by regulator gene.

5. The functioning of operon is stopped when operator gene is covered.

6. Promoter gene is the gene which provides point of attachment to RNA polymerase required for transcription of structural genes.

7. Structural genes are genes which transcribe mRNA for polypeptide synthesis.

8. An operon may have one or more structural genes, e.g., 3 in lac operon, 5 in tryptophan operon, 9 in histidine operon.

9. The polypeptides may become component of structural proteins, enzymes, transport proteins, hormones, antibodies, etc. Some structural genes also form non-coding RNAs.

10. The mechanism of regulation of protein synthesis utilizing operon model can be illustrated using two examples (lac & tryptophan) in bacteria.”

Inducible Operon System (Induction of Operon):

1. Inducible operon system is (a) regulated operon system in which the structural genes remain switched off unless and until an inducer is present in the medium. (Fig. 8.4B)

2. It occurs in catabolic pathways.

3. Lac operon of Escherichia coli is an inducible operon system which was discovered by Jacob and Monod (1961).

4. Lac operon of Escherichia coli has three structural genes, z, y, and a.

5. In the induced operon the structural genes transcribe a polycistronic mRNA which produces three enzymes. These are β-galactosidase, galactoside permease and galactoside acetylase.

6. β-galactoside brings about hydrolysis of lactose or galactoside to form glucose and galactose.

7. Galactoside permease is required for entry of lactose or galactoside into the bacterium.

8. Galactoside acetylase is a transacetylase which can transfer acetyle group to β-galactoside.

9. The initiation codon of structural gene z is TAG (corresponding to AUG of mRNA) and is located 10 base pairs away from the end of the operator gene.

10. The substance whose addition induces the synthesis of enzyme is called inducer.

11. Inducer is a chemical which attaches to repressor and changes the shape of operator binding site so that repressor no more remains attached to operator.

12. In the lac operon allolactose is the actual inducer while lactose is the apparent (visible) inducer.

13. Inducers which induce enzyme synthesis without getting metabolized are called gratuitous inducers, e.g. IPTG (Isopropyl thiogalactoside).

14. Regulator gene (gene) produces mRNA that synthesises a biochemical repressor.

15. Repressor is a small protein formed by regulator gene which binds to operator gene and blocks structural enzyme thus checking mRNA synthesis.

16. The represseor of lac operon is a tetrameric protein having a molecular weight of 1, 60,000. It is made up of 4 subunits each having molecular weight of 40,000.

17. The repressor protein has two sites, a head for attaching to operator gene and a groove for attachment of inducer.

18. Promoter gene functions as a recognition point for RNA polymerase. RNA polymerase initially binds to this gene. It becomes functional only when it is able to pass over the operator gene and reach structural genes.

19. Operator gene controls the expressibility of the operon. It is normally switched off due to binding of repressor over it.

20. However, if the repressor is withdrawn by the inducer, the gene allows RNA polymerase to pass from promoter gene to structural gene.

21. In lac operon the operator gene is small, 27 base pairs long. The gene is made of palindromic or self-complementary sequences.

22. If lactose is added, the repressor is rendered inactive so that it cannot attach on operator gene and synthesis of mRNA takes place.

23. Transcription is under negative control when lac repressor is inactivated by inducer.

24. Transcription in lac operon is under positive control through cyclic AMP receptor protein (CAP).

25. The catabolite gene activator protein (Cga protein) or cyclic AMP receptor protein (CAP) binds to the Cga site.

26. When CAP is attached to the binding site the promoter becomes a stronger one.

27. CAP only attaches to the binding site when bound with cAMP.

28. When glucose level is high cAMP does not occur and so CAP does not bind and hence RNA polymerase do not bind, resulting in low transcription.

29. Lac operon will not however remain operative indefinitely despite presence of lactose in the external environment.

30. It will stop its activity with the accumulation of glucose & galactose in the cell beyond the capacity of the bacterium for their metabolism.

Repressible Operon System (Repression of Operon e.g. Tryptophan Operon of E.coli):

1. A repressible operon system is a regulated segment of genetic material which normally remains operational but can be switched off when its product is either not required or crosses a threshold value.

2. This system is commonly found in anabolic pathways.

3. Tryptophan operon of Escherichia coli is one such repressible operon system. (Fig. 8.5).

4. Tryptophan operon has 5 structural gene – E, D, C, and B A.

5. The gene E and D encodes for enzyme anthranilate synthetase, gene C for glycerol phosphate synthetase, gene B for β subunit of tryptophan synthetize and A for α subunit of tryptophan synthetize.

6. Regulator gene (trp-R) produces a biochemical, generally a proteinaceous substance, called aporepressor.

7. Aporepressor alone is unable to block the operator gene because of the absence of the binding head. Therefore, the operon system remains switched on.

8. A complete repressor is formed only when a non-proteinaceous corepressor joins the aporepressor,

9. Corepressor is a non-proteinaceous component or repressor which is also an end product of reaction catalysed by enzymes produced through the activity of structural genes.

10. It (corepressor) combines with aporepressor and forms repressor which then blocks the operator gene to switch off the operon.

11. The structural genes stop transcription and the phenomenon is known as feed-back repression.

12. Corepressor of tryptophan operon is amino acid tryptophan.

13. In tryptophan the repressor gene is not adjacent to promoter but located in another part of E. coli genome.

16. Promoter gene (trp-P) is the recognition as well as initiation point for RNA polymerase. RNA polymerase attaches to promoter gene. It can pass to structural genes provided the operator gene is in the functional state.

17. Operator gene (trp-O) lies in the passage-way between promoter and structural genes. Normally it remains switched on so that RNA polymerase can pass over from promoter gene to structural gene and bring about transcription.

18. The operator gene can be switched off when both aporepressor and corepressor join together to form repressor. The repressor binds to operator gene to interrupt movement of RNA polymerase.

19. In absence of tryptophan, the RNA polymerase binds to the operator site and thus structural genes are transcribed.

20. The transcription of structural gene leads to the production of enzyme (tryptophan synthetize) that synthesizes tryptophan.

21. When tryptophan becomes available, the enzymes for synthesizing tryptophan are not needed, co-repressor (tryptophan) – repressor complex blocks transcription.

22. One element of tryptophan operon is the leader sequence ‘L’ that is immediately 5′ end of trp. E gene.

23. This ‘L’ sequence controls expression of the operon through a process called attenuation.

24. Attenuation is the termination of the transcription prematurity at the leader region.

25. The tryptophan operon is a negative control.

The two operon models described above can be summarized as given below:

Active Repressor + Operator → System OFF

Active Repressor + Inducer = Inactive Repressor → System ON

(ii) Repressible System:

Apo-repressor and co-repressor complex = Active repressor → System OFF

Apo-repressor = Inactive Repressor → System ON

Importance of Gene Regulation:

1. There are two types of gene action – constitutive and regulated.

2. The constitutive gene action occurs in those systems which operate all the time and the cell cannot live without them, e.g., glycolysis. It does not require repression. Therefore, regulator and operator genes are not associated with it.

3. In regulated gene action all the genes required for a multistep reaction can be switched on or off simultaneously.

4. The genes are switched on or off in response to particular chemicals whether required for metabolism or are formed at the end of a metabolic pathway.

5. Gene regulation is required for growth, division and differentiation of cells. It brings about morphogenesis.


The Yin and Yang of P-TEFb regulation: implications for human immunodeficiency virus gene expression and global control of cell growth and differentiation

The positive transcription elongation factor b (P-TEFb) stimulates transcriptional elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and antagonizing the effects of negative elongation factors. Not only is P-TEFb essential for transcription of the vast majority of cellular genes, but it is also a critical host cellular cofactor for the expression of the human immunodeficiency virus (HIV) type 1 genome. Given its important role in globally affecting transcription, P-TEFb's activity is dynamically controlled by both positive and negative regulators in order to achieve a functional equilibrium in sync with the overall transcriptional demand as well as the proliferative state of cells. Notably, this equilibrium can be shifted toward either the active or inactive state in response to diverse physiological stimuli that can ultimately affect the cellular decision between growth and differentiation. In this review, we examine the mechanisms by which the recently identified positive (the bromodomain protein Brd4) and negative (the noncoding 7SK small nuclear RNA and the HEXIM1 protein) regulators of P-TEFb affect the P-TEFb-dependent transcriptional elongation. We also discuss the consequences of perturbations of the dynamic associations of these regulators with P-TEFb in relation to the pathogenesis and progression of several major human diseases, such as cardiac hypertrophy, breast cancer, and HIV infection.

Figures

P-TEFb phosphorylates the Pol II…

P-TEFb phosphorylates the Pol II CTD and negative elongation factors to stimulate processive…

P-TEFb is essential for Tat…

P-TEFb is essential for Tat transactivation of HIV-1 transcription. Shortly after transcription is…

Domain structure of HEXIM1. The…

Domain structure of HEXIM1. The N-terminal domain of HEXIM1 functions as a self-inhibiting…

Architectural resemblance between the Tat-TAR-P-TEFb…

Architectural resemblance between the Tat-TAR-P-TEFb and HEXIM1-7SK-P-TEFb ribonucleoprotein complexes. (A) Comparison of nucleotide…

P-TEFb is maintained in a functional equilibrium by dynamic associations with its positive…


Activator

One example of an activator is the protein CAP. In the presence of cAMP, CAP binds to the promoter and increases RNA polymerase activity. In the absence of cAMP, CAP does not bind to the promoter. Transcription occurs at a low rate.

Practice Question

What is the role of an activator?

Repressor

When an amino acid is present, it associates with the met repressor, and the repressor is activated. RNA synthesis is blocked by the presence of the repressor on the DNA strand. When the amino acid is not present, the repressor dissociates from the operator and RNA synthesis proceeds.

When tryptophan is not present in the cell, the repressor by itself does not bind to the operator therefore, the operon is active and tryptophan is synthesized. But when a cell has plenty of tryptophan, it doesn’t need to synthesize more. So the repressor is triggered (by the presence of plenty of tryptophan), thus turning off further synthesis of tryptophan.


The Role of Chromatin

Although transcriptional repressors often participate in gene regulation, it must be kept in mind that the very nature of DNA in eukaryotic cells tends to keep genes in the repressed state. Eukaryotic DNA is wrapped around protein complexes called histone octamers, which has the effect of packaging the DNA into a compact form such that it fits inside the nucleus. However, this also limits access of regulatory factors to their target sites. As the mechanisms of transcriptional activators are being uncovered, more and more are being found that act by relieving chromatin-induced repression. An example is the Swi/Snf protein complex, first identified in yeast. Mutations in components of the complex resulted in decreased activity of certain target genes. It was later found that mutations in the histone genes restored normal activity to those target genes in other words, the mutations in the histone genes somehow compensated for the mutations in Swi/Snf. This was an indication that histones and Swi/Snf interact in some way and suggested that Swi/Snf might function by disrupting histone binding to DNA. Biochemical experiments carried out later on showed that this was indeed the case. Although Swi/Snf does not completely dissociate histones from DNA, it loosens them, which is sufficient to allow many activators to bind. Swi/Snf is only involved in activating a subset of genes, and the question of why it functions at some promoters and not others is a topic of intense research.

A second mechanism by which chromatin-induced repression is relieved is by histone acetylation . Histones are positively charged proteins and hence interact tightly with DNA, which is negatively charged. Acetylation of histones reduces their net positive charge, which loosens their interaction with DNA and increases transcription factor binding. Several transcription factors in a variety of organisms have now been found to be acetyltransferases in effect, they can acetylate histones.

In addition, some transcriptional repressors in yeast and mammals have been found to be histone deacetylases. In fact, the protein MeCP2, which binds to methylated DNA, has been found to function in a complex with a histone deacetylase. Thus, methylation would lead to binding of this complex, causing deacetylation of histones and a more condensed chromatin structure. Methylated DNA has long been known to be associated with transcriptionally inactive genes, and inroads into the study of histone acetylation have finally provided an explanation for this.


Author information

These authors contributed equally: Tatsuaki Kurosaki, Maximilian W. Popp

Affiliations

Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY, USA

Tatsuaki Kurosaki, Maximilian W. Popp & Lynne E. Maquat

Center for RNA Biology, University of Rochester, Rochester, NY, USA

Tatsuaki Kurosaki, Maximilian W. Popp & Lynne E. Maquat

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Contributions

All authors contributed equally to the discussion of the content and to the writing and editing of the manuscript before submission.

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Affiliations

Department of Molecular and Cellular Biology, University of California, Davis, CA, 95616, USA

Marissa K Simon, Luis A Williams, Kristina Brady-Passerini, Ryan H Brown & Charles S Gasser

HHMI, Harvard University, Cambridge, MA, 02138, USA

General Mills, Kannapolis, NC, 28081, USA

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