7.2. Negative regulation:
Some protein inhibits the expression of gene by binding on a specific DNA sequence (generally bind with operator), These proteins are called repressor protein. Repressor protein may present active and inactive form.
7.3. Lac operon :
Lac operon (lactose operon) is a best example of operon system. Genes of lac operon encoded enzymes that utilize in conversion of lactose into glucose and galactose. Lac operon consists of following components:
- Lac operator: Operator is the regulatory sequence of gene where repressor protein binds. Lac operon contains three operator, primary operator O1 and two secondary operator - O2 and O3. Primary operator O1 present between promoter and Lac Z gene. O2 located within Lac Z gene and O3 is located at upstream of promoter region. If repressor protein binds with operator, it inhibits binding of RNA polymerase to promoter.
- Promoter: RNA polymerase bound at promoter site.
- Structural gene of Lac operon
Three structural genes are found in Lac operon
- Lac Z: Code for β-galactosidase
- Lac Y: Code for Lactose permease
- Lac A: Code for transacetylase.
β-galactosidase enzyme convert lactose into glucose and galactose. β-galactosidase also initiate process of formation of allolactose (isomer of lactose) from lactose. Lactose permease functions as transporter of lactose from outside to inside the cell.
There is not very important role of transacetylase in lactose metabolism but found to transfer acetyl group from Acetyl CoA to β-galactosidase enzyme and cause the formation of acetyl β-galactosidase, which increases the turn over rate of enzyme. The affinity of β-galactosidase to form glucose and galactose get increase after acetylation. These three gene are transcribed into a single mRNA molecule (polycistronic mRNA) which translated into three different proteins by three independent open reading frame.
- Regulatory gene : A regulatory gene also present in lac operon designated as lac I. This encoded a repressor protein. Lac I gene located at upstream of LacZ, lacY, lac A with their own promoter. Transcription of lac I gene occur in opposite direction of structural gene. Repressor protein consists of four identical subunit.
Repressor protein has helix turn helix motif, for binding with major groove of operator region of DNA. Repressor protein also bind with allolactose through its allolactose binding domain. Allolactose inhibited the binding of repressor protein to operator. In the presence of lactose repressor protein become inactive after binding with allolactose form and in the absence of lactose, repressor become active and bind with operator and block the transcription.
Regulation of Lac operon : Two proteins are responsible for the regulation of lac operon by regulate the activity of promoter that in turn controlling the expression of the lac operon.
One activator that enhances the binding of RNA polymerase to the promoter called positive control.
Second repressor that prevents the RNA polymerase from transcribing called negative control.
7.3.1. Negative control of Lac operon:
Negative control of lac operon by repressor protein, which encoded by Lac I gene. Lac I expression on not regulated. It always express in cells. This type of genes whose expression always occurs called as constitutive gene expression.
If lactose is absent in the cell, repressor protein bind at the operator and stop the transcription. If lactose present in cell, it convert into allolactose by β-galactosidase, allolactose act as inducer and inactivates repressor protein. Inactive repressor cannot bind with operator and now RNA pdymerase effectively bind with promoter and transcribed the gene so gene gets on.
Repressor protein does not bind with operator with high affinity so when repressor protein dissociate from operator. RNA Polymerase bind with promoter and transcribe the genes. This cause minimum or basal level of transcription. This is also called as leaky transcription.
7.3.2. Positive Control of Lac Operon:
In the presence of glucose, E. coli utilize glucose instead lactose, because of glucose is easily metabolized. In the presence of glucose catabolism of other carbohydrate is reduced or suppressed. This is called catabolite repression.
When glucose is present, the level of ATP is increased and level of cAMP is reduced because in the presence of glucose, adenylate cyclase become inactive in medium. Thus ATP not convert into cyclic AMP and activation of cAMPphosphodiesterase also takes place result in decrease in existing cAMP. cAMP is necessary for transcription of lactose operon because cAMP bind with a protein, CAP (catabolite activator protein) or cAMP receptor protein (CRP). CAP is an activator of Lac operon. After binding of cAMP, CAP become active and enhances the binding of RNA polymerase to promoter. But if cAMP level is low, CAP remain inactive and transcription is block.
But in the low concentration of glucose, adenylate cyclase remain active and it convert ATP into cAMP. This cAMP bind with CAP and activate the CAP. Thus cAMP-CAP complex bind with promoter. The cAMP–CRP (CRP in dimer form) complex binds downstream from the promoter and upstream to the transcription start point. cAMP-CRP complex also directly interacts with RNA polymerase and it distorts the DNA by creating 90° band. Thus the RNA polymerase can bind more effectively with promoter and greatly enhanced transcription of the lac operon.
But if glucose concentration is high in medium, glucose come inside the cell through glut transporter (PEP-PTS) an another enzyme EIIB associate with glut transporter. The E II B protein transfer a phosphate to glucose and convert it into glucose-6-phosphate thus during transportation ATP is not require to make glucose-6-phosphat . The glucose-6-phosphateconvert into PEP (phosphoenol pyruvate).
Now PEP change in pyruvate but in this conversion ATP formation does not occur because of PEP transfer its phosphate group to enzyme E1 rather than ADP. E1 transfer phosphate to unphosphorylate Hpr. Hpr transfer phosphate to unphosphorylated EIIA and at last EIIA phosphorylated EIIB. This cycle continuously run till all glucose present outside the cell is not completely utilized.
This cycle is known as PEP-PTS (PEP-dependent phosphotransferase system. Thus EIIA remain unphosphorylated form because of phosphate group not always remain at EIIA, while phosphate remain in flow. Unphosphorylate EIIA block adenylate cyclase and ultimately Lac operon and unphosphorylated EIIA block lactose permease. So in the presence of glucose lac operon became shut down.
7.3.3. Mutational studies of Lac operon
- Mutation in promoter: Transcription is blocked and gene remains in off condition because RNA polymerase can not bind with mutated promoter. This is the type of mutation known as uninducible (induction of expression of structural gene not occur).
- Mutation in operator: When mutation occurs in operator of Lac-operon. The repressor protein not able to bind with mutated operator. The affinity of RNA polymerase for promoter not affected and the genes are transcribed. This type of expression known as constitutive expression.
- Mutation in structural gene: Mutation in structural gene gives non functional product.
- Mutation in repressor gene: Mutated repressor gene (Lac I) encoded non functional repressor protein that cannot bind with operator. Repressor protein is tetrameric protein has four subunit. Repressor protein has three domain.
- DNA binding domain (DBD) bind with DNA.
- Allolactose binding site (ABS): Allolactose bind at allolactose binding site and repressor protein inactive. If mutation in ABS, allolactose (work as an inducer which inactivate the Repressor protein) not able to bind with repressor protein and gene are not expressed. The high concentration of allolactose can turn on the gene, hence called super suppressed mutation. Mutation in ABS noted as I-s.
- Oligomerization domain: It help in tetramer formation repressor protein functions only in tetramer form. Tetramerization occurs on DNA, means tetramerization does not occurs before binding. Repressor protein remains inactive (Repressor protein only functional in tetramer form) and gene gives constitutive expression. Mutation in oligomerization domain denoted as IO.
- If mutation in DBD of repressor protein than gene always in ‘ON’ condition an called so constitutive. Mutation in DBD is noted as I-d .
7.3.4. Mutation study of Lac operon in merozygotes:
Merozyotes are partial diploid cells. In merozygote we identified that mutation in lac operon is recessive or dominant type. Because dominant (type of mutation cannot be restoring in presence of wild type) and recessive (type of mutation can be restoring in presence of wild type) condition occur only diploid cells. Mutation in lac operon is also cis acting (effect gene expression on same DNA strand to which it occur) or trans acting (effect gene expression on different DNA strand).
Mutation in operator:
In merozygote, if E. coli chromosome is mutated at operator. The Repressor protein cannot bind to it, gene expression occur but exogenote contain normal operator, Repressor protein bind on it and inhibit expression of gene and thus cell survive in lactose containing medium. This type of mutation known as dominant, cis and constitutive.
Mutation in Lac I:
Mutation in Lac I gene may have three type mutation in DBD (I-d), mutation in oligomerization domain (IO) and mutation in ABS (I-s)
When mutation occurs in I-d, gene expression in merozygotes it is suppressed because second chromosome has normal Lac I which encode normal Repressor protein which bind on both operator and suppress the expression. This type of mutation is dominant, trans acting and constitutive.
When mutation occur in IO, In this condition E. coli mutated chromosome Lac I product give mutated Repressor protein And other chromosome give normal Repressor protein since tetramerization occur at DNA than probability of tetramerization at DNA from normal Repressor protein subunit is reducing. Hence probability of gene expression is increased. This type of mutation is trans acting and recessive.
When mutation in ABS I-S , In this condition E. coli mutated chromosome Lac I product give mutated Repressor protein at ABS site. And other chromosome give normal Repressor protein at ABS site. This type of mutation known as trans acting, dominant and uninducible. But if high concentration of allolactose can turn on the gene, hence called super suppressed mutation. Hence probability of gene expression is decreased.
Mutation in promoter:
In merozygote, if E. coli chromosome is mutated at promoter. Thus repressor protein cannot bind to it, gene expression does not occur but exogenote contain normal promoter, then RNA polymerase bind on it and the expression of gene occur and thus cell survive in lactose containing medium. This type of mutation known as recessive and cis acting.
Expression of Lac operon in presence of IPTG : IPTG (isopropyl thio β-galactosidase) is analog of allolactose. It bind with allolactose binding site of repressor protein and induced gene expression to inhibit repressor protein. IPTG not metabolized by β-galactosidase, hence at low concentration it induce gene expression, hence called gratuitous inducer.
Identification of expression of Lac operon:
There are certain method to identified that β-galactosidase is synthesize or not by the bacteria.
- X-gal :- X-gal (5 Bromo, 4-chloro, 3 indole β-galactoside) is substrate for β-galactosidase. After degradation X-gal gives blue color. When E. coli colonies give blue colour in the presence of X-gal. It means cell synthesize β-galactosidase. But if colonies does not produce blue colour, it indicates that β-galactosidase is mutated in bacteria.
- ONPG (ortho-nitrophenyl-β-D-galactoside) is also serving as substrate for β-galactosidase. When β-galactosidase degrade ONPG it give yellow colour but less use in comparison to X-gal because harmful for bacteria.
- There is another β-galactosidase noninducing substrate name Phenyl-β-D-galactopyranoside, which give rise to glucose and galactose upon β -galactosidase action. It is not use as a inducer like allolactose in Lac operon. It is responsible for the growth of bacteria and used in selection of mutants of lacZ.
7.4. Tryptophan operon
Tryptophan amino acid synthesize from chorisimic acid in a multistep pathway. Trp operon consist genes that encode enzymes which synthesize tryptophan from chorismic acid. Trp operon contain five structural gene and a regulatory leader. Structural gene involves in tryptophan synthesis and leader sequence involve in control gene expression.
Control of trp operon expression occur at two level, first is repression (repression control the initiation of transcription) and second is attenuation. Attenuation is called pre mature termination of transcription.
Structure of Trp operon: In Trp operon two promoter; Primary Promoter (P1) and Secondary Promoter (P2). Operator (O) is present within primary promoter. Secondary promoter is a weak promoter which enhance the basal level transcription of trp C, B and Trp A genes.
Trp structural gene are trpE, trpD, trpC, trpB and TrpA these five gene present in downstream of trpL sequence called leader sequence. The length of leader sequence is 162 nucleotide comprises four region 1, 2, 3 and 4, which contain 14 codon ORF including two tryptophan codon at the end of region one. Region 1 and 3 has poly U sequence those are complementary to 2 and 4 contain poly A sequence. These complementary sequence help in regulation of transcription.
Trp operon is negative repressible. Trp R encoded a repressor protein which is present in inactive form but after bind with tryptophan it become active and binds on operator and inhibits expression of trp EDCBA gene.
Some other operon also regulates trp operon such as mtr, aroH, aroL. aroH and aroL gene product play role in chorisimic acid synthesis. Mtr gene product is trp specific permease. Apart from Mtr TraB and aroP also code for trp permease. Thus extracellular trp is uptake by this permease.
7.4.1. Trp operon experssion in the absences or presence of tryptophan :
Tryptophan bind with trpR and trp repressor protein become active after binding of tryptophan. Hence tryptophan acts as co repressor. Trp repressor protein and tryptophan complex bind on operator site and prevent the binding of RNA polymerase. Thus trp operon expression is stop. But if trptophan is absent trp repressor remain in inactive form, And now RNA polymerase easily transcribe the trp structural gene.
7.4.2. Regulation of trp operon through attenuation:
Attenuation is the process of premature mRNA termination in the presence of excess amount of tryptophan. The leader sequence transcript has four complementary sequence and these complementary sequence can form stem loop structure. These sequences of leader sequence called attenuator. In attenuator sequence or leader sequence two adjacent tryptophan codon are present. In prokaryotes transcription is coupled with translation. When nascent leader sequence of mRNA is emerged, ribosome is bound on ribosome binding site of leader sequence and initiate translation of leader sequence.
When tryptophan concentration is high in cell than the trp-tRNA to is found in charged form and ribosome is read the trp codon on nascent leader mRNA. Charge tRNA with tryptophan deliver the tryptophan on growing polypeptide just at the end of translation of leader sequence first and ribosome stalled at a stop codon on region one and mask the region second as a result transcription of third and fourth region occur, both region had complement base. Thus region third and region four form hair pin loop structure. This loop structure terminate the transcription.
Thus transcription of structural gene not takes place. A truncated 140 nucleotides trp L transcript is produced when this premature termination or attenuation occurs.
When Trp is absent the availability of charged tRNA with tryptophan is very less. When ribosome translate emerging leader sequence it reach on trp codon (present in leader sequence region one) and stalled on sequence-one because there are no charged tryptophan tRNA is available.
But transcription is continuous and in this condition the region-one not form hair pin loop with region two because the ribosome is pause at region one of leader sequence. Thus the region one is not free and could not pair with region two however the region two can easily pair with region three. This pairing does not terminate the transcription of structural genes of typtophan operon. Thus no termination loop formation occur so transcription could not stop and structured genes transcribed and translate into enzymes which convert chorisimic acid into tryptophan.