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Gene regulation in prokaryotes

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gene regulation in prokaryotes

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Gene regulation in prokaryotes

  1. 1. 2015 Biochemistry of Nucleic Acids Topic: Activity of Gene Regulation in Prokaryotes Submitted to: Dr. JavedIqbal Submitted by: Jannat Iftikhar B11-16 7th semester Department of Botany University of the Punjab Lahore.
  2. 2. 1 Contents 1. Introduction 2. Discovery 3. Gene regulation in prokaryotes 4. Regulation of transcription 5. Principles of Transcriptional Regulation 6. Lac operon  Structure of lac operon  Mechanism  Positive control by CAP-cAMP complex 7. Tryptophan operon  Structure of trp operon  Mechanism  Attenuation  RibosomalProteins Are Translational Repressors of Their Own Synthesis 8. Conclusion 9. References
  3. 3. 2 “Activity of Gene Regulation in prokaryotes” 1. Introduction: Gene is the segment of DNA that controls all traits of organism that may be physical or metabolical. In information encoded in DNA is transcribed into RNA and then translate into proteins. The ability of cell to switch the genes on and off is of fundamental importance because it enables the cell to respond to the changing environment and it is the basis of cell differentiation. So we can say that: “Gene Regulation is a process in which a cell determines which genes it will express and when.” Regulation of gene expression includes a wide range of mechanisms that are used by the cell to increase or decrease the production of specific gene products (protein or RNA). Sophisticated programs of gene expression are widely observed in biology to trigger developmental pathways, respond to environmental stimuli or adapt to new food sources. 2. Discovery: Gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the versatility and adaptability of an organism by allowing the cell to express protein when needed. Although as early as 1951 Barbara McClintock showed interaction between two genetic loci, Activator (Ac) and Dissociator (Ds), in the color formation of maize seeds, the first discovery of a gene regulation system is widely considered to be the identification in 1961 of the lac operon, discovered by Jacques Monod, in which some enzymes involved in lactose metabolism are expressed by the genome of E.coli only in the presence of lactose and absence of glucose. Furthermore, in 1953, Jacques Monod discovered Enzyme Repression. He observed the presence of tryptophan in medium of E.coli which repressed the synthesis of tryptophan synthetase and then further studies showed that all the enzymes, present in tryptophan biosynthetic pathway are simultaneously repressed in the presence of end product. In bacteria such as E.coli, Salmonella and Streptomyces steps controlling genes in biosynthetic pathway are adjacent on their chromosomes ad they can be coordinately repressed. In eukaryotes, related genes are present in genome in scattered form, so coordinate control becomes more difficult to achieve. This
  4. 4. 3 cluster of bacterial genes was first observed by Miloslav Demerec and his colleges in 1956. 3. Gene Regulation in Prokaryotes: The rate of expression of bacterial gene is controlled mainly at level of transcription. Regulation can occur at both the initiation and termination of mRNA synthesis because bacteria obtain their food from the medium that immediately surrounds them. Their regulation mechanisms are designed to adapt quickly to the changing environment. If a gene is not transcribed then the gene product and ultimately the phenotype will not be expressed. In contrast, eukaryotes have much complex and larger genome and cells of higher organisms are surrounded by constant internal milieu. The ability of such cells to respond to harmones and to impulse in nervous system is thus comparatively more important that the ability to respond rapidly in the presence of certain nutrients. (fig.1) Fig.1. Diagram showing at which stages in the DNA-mRNA-protein pathway expression can be controlled
  5. 5. 4 4. Regulation of Transcription: As in prokaryotes gene regulation occur at transcription level, so Transcription of a gene by RNA polymerase can be regulated by at least five mechanisms;  Specificity factors alter the specificity of RNA polymerase for a given promoter or set of promoters, making it more or less likely to bind to them (i.e., sigma factors used in prokaryotic transcription).  Repressors bind to the Operator, coding sequences on the DNA strand that are close to or overlapping the promoter region, impeding RNA polymerase's progress along the strand, thus impeding the expression of the gene. The image to the right demonstrates regulation by a repressor in the lac operon.  General transcription factors position RNA polymerase at the start of a protein-coding sequence and then release the polymerase to transcribe the mRNA.  Activators enhance the interaction between RNA polymerase and a particular promoter, encouraging the expression of the gene. Activators do this by increasing the attraction of RNA polymerase for the promoter, through interactions with subunits of the RNA polymerase or indirectly by changing the structure of the DNA.  Enhancers are sites on the DNA helix that are bound by activators in order to loop the DNA bringing a specific promoter to the initiation complex. Enhancers are much more common in eukaryotes than prokaryotes, where only a few examples exist.  Silencers are regions of DNA sequences that, when bound by particular transcription factors, can silence expression of the gene. Here, we’ll explain two systems i.e, repression and induction by which gene regulation is accomplished in prokaryotes. 5. Principles of Transcriptional Regulation: i. Gene Expression Is Controlled by Regulatory Proteins: Genes are very often controlled by extracellular signals, in the case of bacteria, this typically means molecules present in the growth medium. These signals are communicated to genes by regulatory proteins, which come in two types: positive regulators, or activators; and negative regulators, or repressors. Typically these regulators are DNA binding proteins that recognize specific sites at or near the
  6. 6. 5 genes they control. An activator increases transcription of the regulated gene; repressors decrease or eliminate that transcription. First, RNA polymerase binds to the promoter in a closed complex (in which the DNA strands remain together). The polymerase-promoter complex then undergoes a transition to an open complex in which the DNA at the start site of transcription is unwound and the polymerase is positioned to initiate transcription. This is followed by promoter escape, or clearance, the step in which polymerase leaves the promoter and starts transcribing. ii. Many Promoters Are Regulated by Activators That Help RNA Polymerase Bind DNA and by Repressors That Block That Binding: At many promoters, in the absence of regulatory proteins, RNA polymerase binds only weakly. This is because one or more of the promoter elements are imperfect. When polymerase does occasionally bind, however, it spontaneously undergoes a transition to the open complex and initiates transcription. This gives a low level of constitutive expression called the basal level. Binding of RNA polymerase is the rate limiting step in this case. To control expression from such a promoter, a repressor need only bind to a site overlapping the region bound by polymerase. In that way repressor blocks polymerase binding to the promoter, thereby preventing transcription, although it is important to note that repression can work in other ways as well. The site on DNA where a repressor binds is called an operator. To activate transcription, an activator just helps polymerase bind the promoter. Typically this is achieved as follows: The activator uses one surface to bind to a site on the DNA near the promoter; with another surface, the activator simultaneously interacts with polymerase, bringing the enzyme to the promoter. This mechanism, often called recruitment, is an example of cooperative binding of proteins to DNA. The interactions between the activator and polymerase, and between activator and DNA, serve merely “adhesive” roles: the enzyme is active and the activator simply brings it to the nearby promoter. Once there, it spontaneously isomerizes to the open complex and initiates transcription. The lac genes of E. coli are transcribed from a promoter that is regulated by an activator and a repressor working in the simple ways. (fig.2)
  7. 7. 6 Fig.2. Activation by Recruitment of RNA Polymerase. (a) In the absence of both activator and repressor, RNA polymerase occasionally binds the promoter spontaneously and initiates a low level (basal level) of transcription. (b) Binding of the repressor to the operator sequence blocks binding of RNA polymerase and so inhibits transcription. (c) Recruitment of RNA polymerase by the activator gives high levels of transcription. RNA polymerase is shown recruited in the closed complex. It then spontaneously isomerizes to the open complex and initiates transcription. iii. Some Activators Work by Allostery and Regulate Steps after RNA Polymerase Binding: Not all promoters are limited in the same way. Thus, consider a promoter at the other extreme from that described above. In this case, RNA polymerase binds efficiently unaided and forms a stable closed complex. But that closed complex does not spontaneously undergo transition to the open complex. At this promoter, an activator must stimulate the transition from closed to open complex, since that transition is the rate-limiting step. Activators that stimulate this kind of promoter work by triggering a conformational change in either RNA polymerase or DNA. That is, they
  8. 8. 7 interact with the stable closed complex and induce a conformational change that causes transition to the open complex. This mechanism is an example of allostery. Some promoters are inefficient at more than one step and can be activated by more than one mechanism. Also, repressors can work in ways other than just blocking the binding of RNA polymerase. For example, some repressors inhibit transition to the open complex, or promoter escape. (fig.3) Fig.3. Allosteric Activation of RNA Polymerase. (a) Binding of RNA polymerase to the promoter in a stable closed complex. (b) Activator interacts with polymerase to trigger transition to the open complex and high levels of transcription. 6. The Lac operon: Lac operon is induced 1000 folds by lactose. The cell is an energy efficient unit that makes protein it needs. E. coli has about three thousand protein encoding genes but only a set of them is expressed at any one time. The best illustration of this efficiency is provided by the enzymes involved in the utilization of disaccharide lactose. The synthesis of these enzymes may be induced up to 1000 folds in respond to the addition of lactose to the culture media. Regulation by enzyme induction has been found in many other bacterial systems that degrade sugars, amino acids and lipids. In these systems, the availability of the substrates stimulates the production of enzymes involved in its degradation. Induction is the production of a specific enzyme (or set of enzymes) in response to the presence of a substrate. The lac operon is a inducible system. (fig.4)
  9. 9. 8 Fig.4. Diagram representing the lac operon; regulatory gene, structural genes.  Structure of the lac operon:  The lac operon consists of the three structural genes, a promoter, regulator, terminator, regulator and an operator.  These three structural genes are; lac Z, lac Y and lac A.  Lac Z encode β-galactosidase, an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose.  Lac Y encode β-galactosidase permease, an inner membrane bound symporter that pumps lactose into the cell using a proton gradient.  Lac A encodes β-galactosidase transacetylase, an enzyme that transfer an ecetyl group from acetyl-CoA to β-galactosides.  Only lac Z and lac Y are necessary for lactose catabolism. lac Operon Gene Gene Function I Gene for repressor protein P Promoter O Operator lac Z Gene for ß-galactosidase lac Y Gene for ß-galactoside permease lac A Gene for ß-galactoside transacetylase
  10. 10. 9  Mechanism: The three enzymes required for the utilization of the lactose are β-galactosidase, β-galactosidase pemease and β-galactosidase transacetylase. The synthesis of these enzymes is regulated coordinately as a unit called operon. Operon is the group of the genes that are nest to each other in DNA and that can be controlled in a unified manner. The genes of the structural enzymes are always transcribed together into a single polycistronic lac mRNA which explains why they are always expressed together. The expression of lac operon is regulated by lac repressor. A protein having four identical subunits of 40,000 Dalton each. In an E.coli there are about ten lac repressor molecules which are coded by the regulatory genes. The repressor binds closely and specifically to short DNA segment called “operator”, which is in location very close to the β-galactosidase gene. The affinity of the repressor to bind to the operator is regulated by inducer, a small molecule that can bind to the repressor. The natural inducer of lactose is allolactose, a metabolite of lactose. However, the analogue IPTG (isopropyl thiogalactosidase) is a more powerful inducer that is preferred in the laboratory. Each subunit of the repressor has one binding site for inducer and upon binding it undergoes a conformational change by which it becomes unable to bind to the operator. In this way the presence of the inducer permits transcription of lac operon which is no longer block by the reppressor. The effect of this conformational change is dramatic. While in the absence of lactose, E.coli cells have an average of only three molecule of β-galactosidase enzyme per cell. After induction of lac operon here thousands molecules of β-galactosidase are present in each cell representing 3% of the total proteins. Promoter is the DNA segment to which RNA polymerase binds when initiating transcription. Repressor binds within the promoter and prevents attachment of RNA polymerase. Two sections are particularly important for RNA transcription, first is TATGTTT sequence located 6-12 bases before the transcription starting site, which is conserved in most prokaryotic promoters and second is a region located 35 bases before the beginning of mRNA which is known to be important because mutation within it will severely inhibit lac expression. RNA polymerase binds to a region of about 80 nucleotides of DNA and RNA polymerase binding site, in fact overlaps with the region covered by the repressor. Studies have shown that repressor bind to the operator blocks the binding of RNA polymerase. The way in which the repressor work is very simple. They bind within the promoter and prevents the attachment of RNA polymerase. (fig.5)
  11. 11. 10 Fig.5. lac operon; system turned off in the absence of lactose, and turned on in the presence of lactose  Positive controlby CAP-cAMP complex: cAMP has a regulatory role in bacteria, activating inducible operons at level of transcription. E.coli has cAMP receptor proteins that is able to bind with cAMP with high affinity. This protein is also known as catabolite activator protein (CAP). CAP is a dimeric protein which when complex with the cAMP is able to bind to a specific site within the lac promoter region. RNA polymerase will recognize the lac promoter if only the CAP-cAMP complex is already bound to it. Therefore, in lac operon in addition to the negative control provided by the repressor there is also a positive control provided by the CAP-cAMP complex. The positive control mechanism is also required for the expression of many other inducible proteins, such as those involved in utilization of maltose, galactose and arabinose. However, cAMP is not necessary for the synthesis of the enzymes required for the utilization of glucose as an energy source. This mechanism is highly beneficial in E.coli because its cAMP level varies according to the available food source. Bacteria growing in the presence of glucose have low cAMP content than those growing in poorer energy source such as lactose. When intracellular cAMP level is low i.e, when glucose is available the CAP protein does not bind to the promoter and lac operon will not turn on even in the presence of lactose. As a result E.coli grown in the presence of both glucose and
  12. 12. 11 lactose, it will use only glucose. This makes good sense because glucose is richest and most efficient energy source. This clears that this mechanism of positive control enables the E.coli to adapt more efficiently to changing environment of natural habitat. (Fig.6) Fig.6. outline of lac operon, and positive control regulation by CAP-Camp complex
  13. 13. 12 7. Tryptophan operon: In tryptophan operon, regulation of transcription occur at initiation and termination. In E. coli the five contiguous trp genes encode enzymes that synthesize the amino acid tryptophan. These genes are expressed efficiently only when tryptophan is limiting. (fig.7)  Structure: Fig.7. Structure of trp operon Trp operon gene Gene function trp R Codes for repressor P promoter O Operator, site of attachment of RNA polymerase TrpE Codes for Anthranllate TrpD Codes for Phosphoribosyl anthranllate transferase TrpC Codes for Phosphoribosyl anthranllate isomerase TrpB Codes for Trp β synthatase trpA Codes for Trp α synthatase
  14. 14. 13  Mechanism: The genes are controlled by a repressor, just as the lac genes are, but in this case the ligand that controls the activity of that repressor (tryptophan) acts not as an inducer but as a co-repressor. That is, when tryptophan is present, it binds the trp repressor and induces a conformational change in that protein, enabling it to bind the trp operator and prevent transcription. When the tryptophan concentration is low, the trp repressor is free of its co-repressor and vacates its operator, allowing the synthesis of trp mRNA to commence from the adjacent promoter. Surprisingly, however, once polymerase has initiated a trp mRNA molecule it does not always complete the full transcript. Indeed, most messages are terminated prematurely before they include even the first trp gene (trpE), unless a second and novel device confirms that little tryptophan is available to the cell. Fig.8. trp operon; system turned on when tryptophan is absent, system turned off when tryptophan is present.
  15. 15. 14  Attenuation: This second mechanism overcomes the premature transcription termination, called attenuation. When tryptophan levels are high, RNA polymerase that has initiated transcription pauses at a specific site, and then terminates before getting to trpE. When tryptophan is limiting, however, that termination does not occur and polymerase reads through the trp genes. Attenuation, and the way it is overcome, rely on the close link between transcription and translation in bacteria, and on the ability of RNA to form alternative structures through intramolecular base pairing. The key to understanding attenuation came from examining the sequence of the 5’ end of trp operon mRNA. This analysis revealed that 161 nucleotides of RNA are made from the tryptophan promoter before RNA polymerase encounters the first codon of trpE. Near the end of the sequence, and before trpE, is a transcription terminator, composed of a characteristic hairpin loop in the RNA, followed by eight uridine residues. At this so-called attenuator, RNA synthesis usually stops (and, we might have thought, should always stop), yielding a leader RNA 139 nucleotides long. (fig.9) Fig.9. Trp Operator Leader RNA. Features of the nucleotide sequence of the trp operon leader RNA. Three features of the leader sequence allow the attenuator to be passed by RNA polymerase when the cellular concentration of tryptophan is low. First, there is a second hairpin (besides the terminator hairpin) that can form between regions 1
  16. 16. 15 and 2 of the leader. Second, region 2 also is complementary to region 3; thus, yet another hairpin consisting of regions 2 and 3 can form, and when it does it prevents the terminator hairpin (3, 4) from forming. Third, the leader RNA codes for a short leader peptide of 14 amino acids that is preceded by a strong ribosome binding site. The sequence encoding the leader peptide has a striking feature of two tryptophan codons in a row. Their importance is underscored by corresponding sequences found in similar leader peptides of other operons encoding enzymes that make amino acids. Thus, the leucine operon leader peptide has four adjacent leucine codons, and the histidine operon leader peptide has seven histidine codons in a row. In each case these operons are controlled by attenuation. The function of these codons is to stop a ribosome attempting to translate the leader peptide; thus, when tryptophan is scarce, little charged tryptophan tRNA is available, and the ribosome stalls when it reaches the tryptophan codons. Thus, RNA around the tryptophan codons is within the ribosome and cannot be part of a hairpin loop. A ribosome caught at the tryptophan codons masks region 1, leaving region 2 free to pair with region 3; thus the terminator hairpin (formed by regions 3 and 4) cannot be made, and RNA polymerase passes the attenuator and moves on into the operon, allowing Trp enzyme expression. If, on the other hand, there is enough tryptophan (and therefore enough charged Trp tRNA) for the ribosome to proceed through the tryptophan codons, the ribosome blocks sequence 2 by the time RNA containing regions 3 and 4 has been made. Ribosome blocking region 2 allows formation of the terminator hairpin (from regions 3 and 4), aborting transcription at the end of the leader RNA. The leader peptide itself has no function and is in fact immediately destroyed by cellular proteases. The use of both repression and attenuation to control expression allows a finer tuning of the level of intracellular tryptophan. It provides a two-stage response to progressively more stringent tryptophan starvation—the initial response being the cessation of repressor binding, with greater starvation leading to relaxation of attenuation. But attenuation alone can provide robust regulation: other amino acid operons like his and leu have no repressors; instead, they rely entirely on attenuation for their control. (fig.10)
  17. 17. 16 Fig.10. mechanism of transcriptional attenuation of trp operon  Ribosomal Proteins Are Translational Repressors of Their Own Synthesis: Regulation of translation often works in a manner analogous to transcriptional repression: a “repressor” binds to the translation start site and blocks initiation of that process. In some cases, this binding involves recognition of specific secondary structures in the mRNA. We consider here the regulation of the genes that encode ribosomal proteins. Correct expression of ribosomal protein genes poses an interesting regulatory problem for the cell. Each ribosome contains some 50 distinct proteins that must be made at the same rate. Furthermore, the rate at which a cell makes protein, and thus the number of ribosomes it needs, is tied closely to the cell’s growth rate; a change in growth conditions quickly leads to an increase or decrease in the rate of synthesis of all ribosomal components. Control of ribosomal protein genes is simplified by their organization into several different operons, each containing genes for up to 11 ribosomal proteins. Some non ribosomal proteins that also are required according to growth rate are contained in these operons, including RNA polymerase subunits α, β, and β’. As with other operons, these are sometimes regulated at the level of RNA synthesis. But, the primary control of ribosomal protein synthesis is at the level of translation of the mRNA, not transcription. This distinction is shown by a simple experiment. When extra copies of a ribosomal protein operon are introduced into the cell, the
  18. 18. 17 amount of mRNA increases correspondingly, but synthesis of the proteins stays nearly the same. Thus, the cell compensates for extra mRNA by curtailing its activity as a template. This happens because ribosomal proteins are repressors of their own translation. For each operon, one (or a complex of two) of the ribosomal proteins binds the messenger near the translation initiation sequence of one of the first genes of the operon, preventing ribosomes from binding and initiating translation. Repressing translation of the first gene also prevents expression of some or all of the rest. This strategy is very sensitive. A few unused molecules of protein L4, for example, will shut down synthesis of that protein, as well as synthesis of the other ten ribosomal proteins in its operon. In this way, these proteins are made just at the rate they are needed for assembly into ribosomes. How one protein can function both as a ribosomal component and as a regulator of its own translation is shown by comparing the sites where that protein binds to ribosomal RNA and to its messenger RNA. These sites are similar both in sequence and in secondary structure. The comparison suggests a precise mechanism of regulation. Since the binding site in the messenger includes the initiating AUG, mRNA bound by excess protein S7 cannot attach to ribosomes to initiate translation. (This is analogous to Lac repressor binding to the lac promoter and thereby blocking access to RNA polymerase.) Binding is stronger to ribosomal RNA than to mRNA, so translation is repressed only when all need for the protein in ribosome assembly is satisfied. 8. Conclusion : Trp operon and lac operon are two important biosynthetic systems in E.coli that enable them to adapt quickly according to the changing environment. Any imbalance or mutation at any stage or in any gene effect their activity and ultimately their metabolic functions will be disturbed and they will not be able to cope up with the demands of the environment. Prokaryotes are unicellular or colonial organisms. A number of genes are present in them but different genes in the same cell are activated at different times. This also help the cell to spend a lot of energy to activate different genes that are not required by the cell at particular time. And in prokaryotes regulation is accomplished at transcription level.
  19. 19. 18 9. References  Essentials of molecular biology, George M. Malancinski, 4th edition,2005.  Genetic structure and function, P.F. Smith Keary, 1975,Halsted press,New York.  An introduction to genetic analysis, Grifith AJF,Miller JH, Suzuki DT, et al, 7th edition, 2000.  Genetics by strickberger,3rd edition.  http://en.wikipedia.org/wiki/Trp_operon  http://en.wikipedia.org/wiki/Lac_operon  http://en.wikipedia.org/wiki/Regulation_of_gene_expression  biology.kenyon.edu/courses/biol63/watson_16.pdf

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