Gene Regulation (Lac Operon) PDF

GENE REGULATION ▪ Enzymes and other proteins are required by cells for particular metabolic reactions and in specific concentrations. ▪ Bacterial cells need to control protein production so that there is no wastage of energy, protein or enzymes that are not required by the cell. ▪ Bacterial ce...

GENE REGULATION ▪ Enzymes and other proteins are required by cells for particular metabolic reactions and in specific concentrations. ▪ Bacterial cells need to control protein production so that there is no wastage of energy, protein or enzymes that are not required by the cell. ▪ Bacterial cells achieve this in three different ways; (i)Rapid degradation of mRNA soon after translation. (ii)By allosteric interaction especially with enzymes. (iii)an operon. The operon system (Francois and Monod, 1965) ▪ It is a system of DNA that contains a sequence of genetic code or carries a sequence of genes ▪ These genes code for mRNA that directs the synthesis of enzymes for protein synthesis. ▪ An operon is a coordinated system where all genes coordinate to mediate the regulation of gene expression. ▪ Most common is lac operon ▪ An operon system consists of regulatory elements and structural genes ▪ The structural genes are promoter, operator and regulator. 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 is a region for initiation of transcription. ▪ It is 100 base pairs long. It consists of palindromic sequences. This site promotes and controls the transcription of structural genes or ▪ mRNA. The promoter site is regulated by the regulatory genes of the repressor. 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” which can bind to the promoter and operator. Structural elements of the lac operon ▪ 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 (Galactoside permease). Function: Lactose permease brings lactose into the cell. ▪ Lac-A: Encodes for the enzyme thiogalactoside transacetylase also called thiogalactoside transacetylase (GAT). Function : transfers an acetyl group from acetyl-CoA to galactosides, glucosides and lactosides. It is coded for by the lacA gene of the lac operon in E. coli. Structure of the lac operon The Lac Operon ▪ The lac operon is a bio-synthesis system that is required for the efficient transport and metabolism of lactose (milk sugar) in Escherichia Coli and other related bacteria. ▪ In the absence of glucose, the E. coli bacteria cells can utilise lactose as a source of energy however it has to be processed via the lac operon. ▪ When there is no lactose (diagram 1 on next slide), the lac repressor binds tightly to the operator. It gets in RNA Polymerase’s way, thereby preventing transcription. ▪ With lactose (diagram 2,) allolactose (rearranged lactose) binds to the lac repressor and makes it let go of the Operator. RNA polymerase can now transcribe the operon. They are activated by a single promoter and produce a single mRNA molecule. ▪ The same sequence of nucleotide bases which constitute an operon also carry non structural genes that do not code for protein but aid the operon in regulating the metabolism of lactose. ▪ These extra genes on the operon are collectively called regulator sites. ▪ Specifically they are a promoter site, a terminator site and an operator site. ▪ Therefore, a lac operon is a sequence of nucleotide bases divided into three structural genes and three regulator sites for metabolism of lactose. ▪ Specific control of the lac genes depends on the presence or absence of lactose in a cell. ▪ When lactose is absent in the in the bacteria growth, the lac gene remain dormant because the need to produce enzymes required to process the lactose does not arise. ▪ When lactose is present enzyme β-galactoside permease insert the membrane and allows lactose to flood into the cell at a faster rate. ▪ Once the lactose is inside the cell, enzyme β-galactosidase breaks down lactose into simpler sugars: glucose and galactose. ▪ These simple sugars are the used as source of energy by the cells. ▪ Enzyme β-galactoside transacetylase facilitates the transfer of acetyl group from acetyl-CoA to β-galactoside. The lac repressor protein finally rolls out ▪ Since there is no more interference along the lac operon, RNA polymerase can attaches to the promoter site and transcribe the lac operon genes on to the mRNA ▪ This happens as mRNA moves along the DNA from 3’ to 5’. ▪ As the lactose substrate runs out in the medium, its absence induces a repressor protein to go back to the original shape and bind to the DNA operator site once again. Control of gene expression in prokaryotes ▪ Controlled in two ways: positive control and negative control Positive inducible system. It includes the following steps: 1. The regulatory gene is expressed by the repressor. 2. After expression of a regulatory gene, the repressor produces repressor proteins 3. Repressor protein has binding sites for the operator and the inducer i.e. lactose. 4. Therefore, when lactose is present (inducer) it binds with the repressor protein and forms “R+I complex”. 5. After the binding of the inducer to the repressor, it 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 inducer). Diagrammatic representation of positive control Negative control of the lac operon It includes the following steps: 1. First, the regulatory gene is expressed by the repressor. 2. After expression of a regulatory gene, the repressor proteins produces a repressor protein. 3. In the absence of the inducer (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, inhibits the mRNA transcription. ▪ This concept is known as “switch off” of Lac-operon (by the absence of inducer). Diagrammatic representation of negative control of repressor system. The inducer (antirepressor) ▪ It suppresses the activity and binding of the repressor protein to the operator and makes it “inactive repressor” from the active repressor. ▪ In the Lac-operon, lactose or allolactose acts as an inducer. Another inducer of the lac operon is isopropylthiogalactoside (IPTG). ▪ Allolactose is formed by the enzyme beta-galactosidase as a result of isomerization of lactose i.e. galactose links to the C6 instead of C4. Inducers and the induction of the lac operon ▪ Normally, E. coli cells make very little of any of these three proteins in the absence of lactose. ▪ When lactose is available there is a large increase in the amount of each enzyme. ▪ Thus each enzyme is an inducible enzyme and the process is called induction. ▪ The mechanism is that the few molecules of ß-galactosidase in the cell before induction convert the lactose to allolactose which then turns on the transcription of these three genes in the lac operon. Lac operon in the absence of inducers ▪ In the absence of an inducer such as allolactose or IPTG, the Lac I gene is transcribed ▪ The resulting repressor protein binds to the operator site of the lac operon, LacO, and prevents transcription of the lacZ, lacY and lacA genes. Lac operon in the presence of inducers ▪ During induction, the inducer binds to the repressor. ▪ This causes a change in the conformation of the repressor that greatly reduces its affinity for the lac operator site. ▪ The lac repressor now dissociates from the operator site and allows the RNA polymerase (already in place on the adjacent promoter site) to begin transcribing the lacZ, lacY and lacA genes. ▪ They are transcribed to yield a single polycistronic mRNA that is then translated to produce all three enzymes in large amounts. ▪ The existence of a polycistronic mRNA ensures that the amounts of all three gene products are regulated co-ordinatelly. ▪ If the inducer is removed, the lac repressor rapidly binds to the lac operator site and transcription is inhibited almost immediately. CRP/CAP ▪ High-level transcription of the lac operon requires the presence of a specific activator protein called catabolite activator protein (CAP), also called (cyclic adenosine monophosphate (cAMP) receptor protein (CRP). ▪ This protein, cannot bind to DNA unless it is complexed with 3’5′ cyclic AMP (cAMP). ▪ The CRP binds to the lac promoter just upstream from the binding site for RNA polymerase. ▪ It increases the binding of RNA polymerase and so stimulates transcription of the lac operon. ▪ Whether or not the CRP protein is able to bind to the lac promoter depends on the carbon source available to the bacterium. Lac operon in the presence of glucose ▪ When glucose is present, E. coli does not need to use lactose as a carbon source and so the lac operon does not need to be active. ▪ Thus the system has evolved to be responsive to glucose. ▪ Glucose inhibits adenylate cyclase, the enzyme that synthesizes cAMP from ATP. ▪ Thus, in the presence of glucose the intracellular level of cAMP falls, so CRP cannot bind to the lac promoter, and the lac operon is only weakly active (even in the presence of lactose). Lac operon in the absence of glucose ▪ When glucose is absent, adenylate cyclase is not inhibited, the level of intracellular cAMP rises and binds to CRP. ▪ Therefore, when glucose is absent but lactose is present, the CRP– cAMP complex stimulates transcription of the lac operon and allows the lactose to be used as an alternative carbon source. ▪ In the absence of lactose, the lac repressor, ensures that the lac operon remains inactive. ▪ These combined controls ensure that the lacZ, lacY and lacA genes are transcribed strongly only if glucose is absent and lactose is present.  Regulation that the lac operon undergoes is termed negative inducible because gene is turned off by The regulator factor (lac repressor). Regulator gene produces a repressor molecule which in the absence of lactose, inhibits the structural genes directly but acts through the operator gene. In eukaryotes gene regulation may occur when DNA is uncoiled & loosened from the nucleosomes (structural unit of a eukaryotic chromosome consisting of a length of DNA coiled around a core of histones) to bind to transcriptional factors (epigenetic level), when the RNA is transcribed (transcriptional level), when RNA is processed and exported to the cytoplasm after it is transcribed (post-transcriptional level), when RNA is translated into protein (translational level), or after the protein has been made (post- translational level). In prokaryotes, RNA transcription and protein translation occur simultaneously. Gene expression in prokaryotes is regulated at the transcriptional level. Genetic code ▪ Each cell in an organism contains all the information required to determine all the characteristics of the whole organism. ▪ This information is called the genetic code ▪ Genetic code is important in knowing how genes function. ▪ Useful in genetic engineering and biotechnology ▪ Sections of DNA called cistrons (genes) contain information needed to make a particular polypeptide ▪ When DNA in a cistron is activated, the information is transferred to mRNA which acts as a template for The synthesis of the polypeptide ▪ Relationship between DNA, mRNA and polypeptides in an eukaryotic cell is called the central dogma of biology. ▪ mRNA is made on a DNA template in a process called transcription. ▪ Gene expression takes place when information in a cistron is used to make a functional polypeptide by transcription and translation. ▪ mRNA is a large polypeptide polymer, chemically similar to DNA but differing in that: mRNA consists of only one chain of nucleotides, mRNA consists of the sugar ribose instead of deoxyribose and mRNA contains the base uracil instead of thymine. The triplet code ▪ 20 amino acids make all proteins in living organisms. If a code consisted of one base for one amino acid, only four combinations would be provided. ▪ If 2 bases coded for one amino acid, there would be 16 (4^2) possible combinations. A three base Code provides 64 (4^3) possible combinations, more than enough for all 20 amino acids ▪ Adding or removing one or two bases causes a frame shift mutation which inactivates a gene. ▪ The genetic code is non-overlapping: each triplet in DNA specifies one amino acid ▪ Each base is part of only one triplet and therefore involved in specifying only one amino acid. ▪ A non-overlapping code requires a longer sequence of bases than an overlapping one. The main features of the genetic code are given below: ▪Degenerate code: there are more codons than amino acids ▪ Triplet code of 3 nucleotides: each of the 20 amino acids used to make proteins is represented by a 3 letter abbreviation (a base triplet) in a DNA or a codon in mRNA ▪ Linear code: reads from a starting point to a finishing point. The codon is always read in the 5’ to the 3’ direction ▪ Punctuation codons: the start and end of a sequence in a cistron is determined by specific codons; the ‘start’ signal is given by AUG, which is methionine. There are 3 ‘stop’ signals (UAA,UAG and UGA) ▪ Almost universal: Most organisms share the same code; chloroplast and mitochondrial DNA have a slightly modified code. (a codon is a sequence of 3 DNA/RNA nucleotides with a specific amino acid or stop signal during protein synthesis)