Different kinds of cellsDifferent kinds of cells

C0NTROL OF GENE C0NTROL OF GENE EXPRESSIONEXPRESSION

• Control of gene expression is Control of gene expression is essential to all organisms: in essential to all organisms: in bacteria it allows the cell to take bacteria it allows the cell to take advantage of changing advantage of changing environmental conditions, in environmental conditions, in multicellular organisms it is critical multicellular organisms it is critical for directing development and for directing development and maintaining homeostasis.maintaining homeostasis.

• One way to control gene expression is to regulate One way to control gene expression is to regulate initiation of transcription. In order for a gene to be initiation of transcription. In order for a gene to be transcribed, RNA-polymerase must have access to the transcribed, RNA-polymerase must have access to the DNA-helix and must be capable of binding to the gene’s DNA-helix and must be capable of binding to the gene’s promoter, a specific sequence of nucleotides at one end promoter, a specific sequence of nucleotides at one end of the gene that tells the polymerase where to begin of the gene that tells the polymerase where to begin transcribing. How is the initiation of transcription transcribing. How is the initiation of transcription regulated?regulated?

• Protein- binding nucleotide sequences on the Protein- binding nucleotide sequences on the DNA regulate the initiation of transcription by DNA regulate the initiation of transcription by modulating the ability of RNA-polymerase to bind modulating the ability of RNA-polymerase to bind to the promoter. This protein- binding sites are to the promoter. This protein- binding sites are usually 10-15 nucleotides in length. Hundreds of usually 10-15 nucleotides in length. Hundreds of these sequences have been characterized and these sequences have been characterized and each provides a specific binding site for a specific each provides a specific binding site for a specific protein able to recognize the sequence. Binding protein able to recognize the sequence. Binding of protein to the regulatory sequence either of protein to the regulatory sequence either blocks transcription by getting the way RNA-blocks transcription by getting the way RNA-polymerase, polymerase, ооr stimulates transcription by r stimulates transcription by facilitating the binding of RNA-polymerase to the facilitating the binding of RNA-polymerase to the promoter.promoter.

Regulation of gene Regulation of gene expression in Prokaryotesexpression in Prokaryotes

• The Operon model for the regulation of gene The Operon model for the regulation of gene expression.expression.

• In 1961 F. Jacob and J. Monod at the Pasteur In 1961 F. Jacob and J. Monod at the Pasteur Institute in Paris proposed that metabolic Institute in Paris proposed that metabolic pathways are regulated as a unit. They were pathways are regulated as a unit. They were awarded Nobel prize in 1965. Based on awarded Nobel prize in 1965. Based on genetic studies of the production of the genetic studies of the production of the enzymes involved in lactose metabolism enzymes involved in lactose metabolism Jacob and Monod proposed the operon Jacob and Monod proposed the operon model to explain gene induction in model to explain gene induction in prokaryotes. Although there are many prokaryotes. Although there are many operons in bacterial cells, the lactose operons in bacterial cells, the lactose (lac.operon) discovered by Jacob and Monod (lac.operon) discovered by Jacob and Monod is the classic example of all operonsis the classic example of all operons

Lactose metabolism in Lactose metabolism in Escherichia coli.Escherichia coli.

• In the absence of glucose, E.coli can use In the absence of glucose, E.coli can use lactose, if present as a source of carbon and lactose, if present as a source of carbon and energy.energy.

• β-Galactosidase is the key enzyme in the β-Galactosidase is the key enzyme in the metabolism of lactosemetabolism of lactose

• by E.coli. It catalyzes the following reaction:by E.coli. It catalyzes the following reaction:• Lactose + H2 O > Galactose + GlucoseLactose + H2 O > Galactose + Glucose• In the absence of lactose, there are fewer In the absence of lactose, there are fewer

than 10 molecules of β-galactosidase per cellthan 10 molecules of β-galactosidase per cell• In the presence of lactose, and no other In the presence of lactose, and no other

energy source, the number of β-galactosidase energy source, the number of β-galactosidase molecules can increase to 5000 molecules per molecules can increase to 5000 molecules per cell within several minutes.cell within several minutes.

• When the sugar lactose is added to the When the sugar lactose is added to the cultures of E.coli it induces three cultures of E.coli it induces three enzymes necessary toenzymes necessary to

• break down the lactose into glucose and break down the lactose into glucose and galactose. These enzymes are galactose. These enzymes are synthesized together in a coordinated synthesized together in a coordinated manner and the unit is called lac operon. manner and the unit is called lac operon.

• Since the addition of lactose itself Since the addition of lactose itself stimulates the production the enzymes it stimulates the production the enzymes it is also known as inducible system.is also known as inducible system.

Lactose (lac) operon.Lactose (lac) operon.

• An operon is a group of coordinately An operon is a group of coordinately regulated genes, theregulated genes, the

• products of which typically catalyze a products of which typically catalyze a multienzyme metabolicmultienzyme metabolic

• pathway and their contralling elements. pathway and their contralling elements. The purpose of the lacThe purpose of the lac

• operon is to make the enzymes operon is to make the enzymes necessary to metabolize lactosenecessary to metabolize lactose

• (i.e., β-galatozidase, galatoside permease, (i.e., β-galatozidase, galatoside permease, thiogalactosidase transacetylase)thiogalactosidase transacetylase)

Two classes ofTwo classes ofgenes are needed to make a functional operon: genes are needed to make a functional operon:

structural genesstructural genes and and regulatory genes.regulatory genes.

• a. The products of operons are produced by a. The products of operons are produced by structural genesstructural genes . .

• Structural genesStructural genes code for products that may be code for products that may be enzyme orenzyme or

• transfer RNA (t-RNA), ribosomal RNA (rRNA), or transfer RNA (t-RNA), ribosomal RNA (rRNA), or ribosomal protein. Structural gene products are ribosomal protein. Structural gene products are essential for the life of the cell.essential for the life of the cell.

• b. b. Regulatory genesRegulatory genes code for products that code for products that regulate the levelregulate the level

• of expression of structural genes. Although of expression of structural genes. Although regulatory genes areregulatory genes are

• often not considered part of operons because they can often not considered part of operons because they can be located at sites remote from structural genes they be located at sites remote from structural genes they regulate, they are key elements of operons.regulate, they are key elements of operons.

Structure of the lac- Structure of the lac- operon.operon.

• The lac operon is a region of DNA in the The lac operon is a region of DNA in the genome that containsgenome that contains

• the following.the following.• Three linked structural genes.Three linked structural genes.• 1. The lac Z gene that codes for B-1. The lac Z gene that codes for B-

galactosidasegalactosidase• 2. The lac Y gene that codes for galactoside 2. The lac Y gene that codes for galactoside

permeasepermease• 3. The lac A gene that codes for 3. The lac A gene that codes for

thiogalactosidase transacetylase.thiogalactosidase transacetylase.

Structure of the lac- Structure of the lac- operon.operon.

• A single promoter directs proper A single promoter directs proper initiation of transcription.initiation of transcription.

• The lac Z, lac Y, and lac A genes are The lac Z, lac Y, and lac A genes are expressed as a polycistronic message expressed as a polycistronic message from this common promoter.from this common promoter.

• An operator region lies adjacent to the An operator region lies adjacent to the promoter and spans the transcriptional promoter and spans the transcriptional initiation site. A regulatory protein called initiation site. A regulatory protein called thethe

• lac repressor binds to this site and lac repressor binds to this site and blocks initiation ofblocks initiation of

• transcription.transcription.

Regulation of lac operon Regulation of lac operon expressionexpression

Negative regulation by the lac Negative regulation by the lac repressor.repressor.

• In the absence of lactose, the cell has In the absence of lactose, the cell has no need for the production of B-no need for the production of B-galactosidase and galactoside galactosidase and galactoside permease. permease.

• A regulatory molecule, the lac repressor, A regulatory molecule, the lac repressor, prevents expression of the lac operon in prevents expression of the lac operon in the absence of lactose. This is the the absence of lactose. This is the example of negative regulation.example of negative regulation.

Structure of the lac- Structure of the lac- repressor.repressor.

• The lac- repressor is a tetrameric The lac- repressor is a tetrameric protein, with each subunit having a protein, with each subunit having a binding site for an inducer. The lac - binding site for an inducer. The lac - repressor is a diffusible product of the repressor is a diffusible product of the regulatory lac I gene. regulatory lac I gene.

• The lac I – gene is adjacent to the lac The lac I – gene is adjacent to the lac operon. operon.

• The lac I gene maintains the very low The lac I gene maintains the very low level of lac - repressor needed for the level of lac - repressor needed for the regulation of the lac-operon.regulation of the lac-operon.

• In the absence of inducer, the lac In the absence of inducer, the lac repressor binds tightly to the repressor binds tightly to the operator, which blocks initiation of operator, which blocks initiation of transcription of the structural transcription of the structural genes. genes.

• The presence of inducer (allolactose) The presence of inducer (allolactose) relievesrelieves

• the negative regulation by the lac the negative regulation by the lac repressor. Upon binding of the inducer to repressor. Upon binding of the inducer to the lac repressor, the repressor the lac repressor, the repressor undergoes a conformational change to undergoes a conformational change to a shape that no longer binds the a shape that no longer binds the operator tightly. With the repressor no operator tightly. With the repressor no longer blocking the initiation site RNA longer blocking the initiation site RNA polymerase initiates transcription.polymerase initiates transcription.

• Since prokaryotes do not have a nucleus, Since prokaryotes do not have a nucleus, there is no physical structure such as a there is no physical structure such as a nuclear membrane to separate nuclear membrane to separate translation, therefore trancription and translation, therefore trancription and translation are coupled in translation are coupled in prokaryotes. Ribosomes bind to the prokaryotes. Ribosomes bind to the polycistronic lac messenger RNA (lac- polycistronic lac messenger RNA (lac- mRNA) and initiate translation even mRNA) and initiate translation even before transcription of lac operon is before transcription of lac operon is complete.complete.

Structure of the lac- Structure of the lac- operonoperon

• As long as lactose is present, then inducer present, As long as lactose is present, then inducer present, andand

• transcription of the lac operon continues. The result transcription of the lac operon continues. The result is theis the

• continued production of the enzymes needed for the continued production of the enzymes needed for the metabolism ofmetabolism of

• lactose.lactose.• After the inducer is removed, expression of the lac After the inducer is removed, expression of the lac

operonoperon• stops quickly. This occurs due to the fact that as with stops quickly. This occurs due to the fact that as with

most most• prokaryotic m-RNAs, the lac m-RNA is unstable and prokaryotic m-RNAs, the lac m-RNA is unstable and

decays withindecays within• minutes.minutes.

The Need for Gene The Need for Gene Regulation in EukaryotesRegulation in Eukaryotes

• While eukaryotes can respond to their While eukaryotes can respond to their environment , the main reason higher environment , the main reason higher eukaryotes need to regulate their genes is cell eukaryotes need to regulate their genes is cell specialization. Whereas prokaryotes are simple, specialization. Whereas prokaryotes are simple, unicellular organisms, multicellular eukaryotes unicellular organisms, multicellular eukaryotes consist of hundreds of different cell types, each consist of hundreds of different cell types, each differentiated to serve a different specialized differentiated to serve a different specialized function. Each cell type differentiates by function. Each cell type differentiates by activating a different subset of genes. Because activating a different subset of genes. Because of the multitude of cell types, the regulation of of the multitude of cell types, the regulation of gene expression required to bring about such gene expression required to bring about such differentiation is necessarily complex. One way differentiation is necessarily complex. One way this complexity is demonstrated is in multiple this complexity is demonstrated is in multiple levels of regulation of gene expression.levels of regulation of gene expression.

Levels of RegulationLevels of Regulation

• Eukaryotic cells are more complex Eukaryotic cells are more complex than prokaryotic cells. than prokaryotic cells.

• Nucleus in eukaryotic cells separates Nucleus in eukaryotic cells separates transcription from translation in a way transcription from translation in a way not seen in prokaryotes.not seen in prokaryotes.

• Eukaryotic transcripts must be Eukaryotic transcripts must be processed before they can be processed before they can be translated. Here is a diagram outlining translated. Here is a diagram outlining the steps involved in the production of the steps involved in the production of a protein in eukaryotic cells:a protein in eukaryotic cells:

Diagram outlining the steps involved in the Diagram outlining the steps involved in the production of a protein in eukaryotic cells:production of a protein in eukaryotic cells:

• Regulation can occur at any point in Regulation can occur at any point in this pathway; specifically, it occurs this pathway; specifically, it occurs at the levels of transcription, RNA at the levels of transcription, RNA processing, mRNA lifetime processing, mRNA lifetime (longevity), and translation. Each of (longevity), and translation. Each of these types of regulation will be these types of regulation will be considered in turn.considered in turn.

Regulation of RNA Regulation of RNA Processing Processing

• After transcription, the RNA must be After transcription, the RNA must be processed before it can be translated. RNA processed before it can be translated. RNA processing involves addition of a 5' cap, processing involves addition of a 5' cap, addition of a 3' poly (A) tail, and removal of addition of a 3' poly (A) tail, and removal of introns. This processing represents another introns. This processing represents another level of regulation of gene expression, level of regulation of gene expression, particularly in regard to splicing out of particularly in regard to splicing out of introns. Regulation can be of two types: a) introns. Regulation can be of two types: a) whether an RNA gets processed; and b) whether an RNA gets processed; and b) which exons are retained in the mRNA. which exons are retained in the mRNA.

• The first type of regulation can determine The first type of regulation can determine whether or not an mRNA gets translated. If an whether or not an mRNA gets translated. If an RNA is not processed, it will not be transported RNA is not processed, it will not be transported out of the nucleus, and will not be translated.out of the nucleus, and will not be translated.

• The second type of regulation can affect the The second type of regulation can affect the function of the protein produced. Some genes function of the protein produced. Some genes have exons that can be exchanged in a process have exons that can be exchanged in a process known as known as exon shufflingexon shuffling. For example, a gene . For example, a gene with four exons might be spliced differently in with four exons might be spliced differently in two different cell types. In cell 1, exons 1, 2, two different cell types. In cell 1, exons 1, 2, and 4 would be used in the mRNA:and 4 would be used in the mRNA:

• . In cell 1, exons 1, 2, and 4 would be used . In cell 1, exons 1, 2, and 4 would be used in the mRNA:in the mRNA:

• In cell 2 on the other hand, exons 1, 3, In cell 2 on the other hand, exons 1, 3, and 4 would be used:and 4 would be used:

In cell 2 on the other hand, exons 1, 3, In cell 2 on the other hand, exons 1, 3, and 4 would be used:and 4 would be used:

• In each of these cases, the polypeptide In each of these cases, the polypeptide produced could have a different function. produced could have a different function. In mammals, for example, the calcitonin In mammals, for example, the calcitonin gene produces a hormone in one cell gene produces a hormone in one cell type, and a neurotransmitter in another type, and a neurotransmitter in another cell type, due to exon shuffling. In cell type, due to exon shuffling. In DrosophilaDrosophila, alternate splicing of the , alternate splicing of the sex-sex-lethallethal RNA can produce an mRNA RNA can produce an mRNA encoding a functional polypeptide, or one encoding a functional polypeptide, or one with a premature stop codon that encodes with a premature stop codon that encodes a short, nonfunctional polypeptide.a short, nonfunctional polypeptide.

Regulation of RNA Regulation of RNA LongevityLongevity

• Imagine two mRNA molecules: one lasts for five Imagine two mRNA molecules: one lasts for five minutes in the cytoplasm before being degraded, while minutes in the cytoplasm before being degraded, while the other one manages to linger for an hour before the other one manages to linger for an hour before being degraded. If both are translated continually while being degraded. If both are translated continually while they exist, it is obvious that more of the second they exist, it is obvious that more of the second polypeptide will be produced than the first. This is the polypeptide will be produced than the first. This is the principle behind regulation of RNA longevity. mRNAs principle behind regulation of RNA longevity. mRNAs from different genes have their approximate lifespan from different genes have their approximate lifespan encoded in them; this serves to help regulate how much encoded in them; this serves to help regulate how much of each polypeptide is produced. The information for of each polypeptide is produced. The information for lifespan is found in the 3' UTR. The sequence AUUUA, lifespan is found in the 3' UTR. The sequence AUUUA, when found in the 3' UTR, is a signal for early when found in the 3' UTR, is a signal for early degradation (and therefore short lifetime). The more degradation (and therefore short lifetime). The more times the sequence is present, the shorter the lifespan times the sequence is present, the shorter the lifespan of the mRNA. Because it is encoded in the nucleotide of the mRNA. Because it is encoded in the nucleotide sequence, this is a set property of each different sequence, this is a set property of each different mRNA; the longevity of an mRNA can't be varied.mRNA; the longevity of an mRNA can't be varied.

Regulation of Regulation of TranslationTranslation

• Whether or not an mRNA molecule is Whether or not an mRNA molecule is translated can be regulated as well. The translated can be regulated as well. The various mechanisms of translational regulation various mechanisms of translational regulation are incompletely understood, but there are are incompletely understood, but there are many documented examples (particularly in many documented examples (particularly in embryonic development) of mRNA molecules embryonic development) of mRNA molecules that are present routinely, but are only that are present routinely, but are only translated under certain circumstances. For translated under certain circumstances. For example, many animals sequester large example, many animals sequester large amounts of mRNA in their eggs, and those amounts of mRNA in their eggs, and those mRNA molecules are not translated unless the mRNA molecules are not translated unless the egg is fertilized.egg is fertilized.

Regulation of TranscriptionRegulation of Transcription• Many different proteins participate in Many different proteins participate in

transcription. Some of these are required for transcription. Some of these are required for the transcription of all structural genes and are the transcription of all structural genes and are termed general transcription factors. Others, termed general transcription factors. Others, labeled specific transcription factors, have labeled specific transcription factors, have more specialized roles, activating only certain more specialized roles, activating only certain genes at certain stages of developmentgenes at certain stages of development A key A key transcriptional element is RNA-polymerase. It transcriptional element is RNA-polymerase. It plays a vital role in initiating transcription by plays a vital role in initiating transcription by binding to the promoter region, but it can not binding to the promoter region, but it can not locate the promoter region on its own. Further locate the promoter region on its own. Further more, it is incapable of producing significant more, it is incapable of producing significant quantities of m-RNA by itself. quantities of m-RNA by itself.

• Effective transcription requires the Effective transcription requires the interaction of a large complex of 50 interaction of a large complex of 50 or so different proteins. These or so different proteins. These include different transcription include different transcription factors, which bind to RNA-factors, which bind to RNA-polymrase and to specific DNA polymrase and to specific DNA sequences in the promoter region sequences in the promoter region ( sequences such as TATA and ( sequences such as TATA and others needed for transcription others needed for transcription initiation). initiation).

• The general transcription factors allow The general transcription factors allow RNA-polymerase to bind to the promoter RNA-polymerase to bind to the promoter region so that it can function effectively region so that it can function effectively in transcription. The transcriptional in transcription. The transcriptional activity of specific genes can be greatly activity of specific genes can be greatly increased by interaction with sequences increased by interaction with sequences called enhancers, which may be located called enhancers, which may be located thousands of bases upstream or thousands of bases upstream or downstream of the genedownstream of the gene A very large A very large number of enhancer elements has been number of enhancer elements has been identified and characterized, and each identified and characterized, and each different enhancer has its own different enhancer has its own transcription factor that it binds to.transcription factor that it binds to.

• Enhancers however, do not interact Enhancers however, do not interact directly with genes. Instead, they are directly with genes. Instead, they are bound by specific transcription bound by specific transcription factors, co-activators, which in turn factors, co-activators, which in turn bind to general transcription bind to general transcription complex. This chain of interactions complex. This chain of interactions from enhancer to activator, to from enhancer to activator, to coactivator, to general transcription coactivator, to general transcription complex and finally to the gene itself, complex and finally to the gene itself, increases the activity of specific increases the activity of specific genes at specific points in time. genes at specific points in time.

• Transcription factors can be activated by signals Transcription factors can be activated by signals from other cells in the same organism. Such signals from other cells in the same organism. Such signals include include hormoneshormones and and growth factorsgrowth factors. Hormones . Hormones must bind to a specific receptor on the target cell, must bind to a specific receptor on the target cell, and the receptor mediates the cellular effects of the and the receptor mediates the cellular effects of the signal. There are two basic mechanisms used, one signal. There are two basic mechanisms used, one for steroid hormones, and one for peptide hormones: for steroid hormones, and one for peptide hormones: – Steroid hormones are lipid (actually cholesterol) Steroid hormones are lipid (actually cholesterol)

derivatives, such as testosterone and progesterone. These derivatives, such as testosterone and progesterone. These hormones can cross the cytoplasmic membrane into a cell, hormones can cross the cytoplasmic membrane into a cell, where they bind to their specific receptor. Steroid receptors where they bind to their specific receptor. Steroid receptors are transcription factors, and when they bind to their are transcription factors, and when they bind to their ligand, they become activated and initiate transcription of a ligand, they become activated and initiate transcription of a specific set of genes. specific set of genes.

– Peptide hormones cannot cross the cytoplasmic membrane, Peptide hormones cannot cross the cytoplasmic membrane, and so must bind to a receptor on the cell surface. When and so must bind to a receptor on the cell surface. When bound to its ligand, these receptors initiate a series of bound to its ligand, these receptors initiate a series of biochemical reactions inside the cell, with the ultimate biochemical reactions inside the cell, with the ultimate result being the activation of a transcription factor (often by result being the activation of a transcription factor (often by phosphorylation of the transcription factor), which initiates phosphorylation of the transcription factor), which initiates transcription of a specific set of genes. transcription of a specific set of genes.

• Transcription factors can be activated Transcription factors can be activated by environmental signals. For example, by environmental signals. For example, virtually all organisms have a set of virtually all organisms have a set of genes called genes called heat shock genesheat shock genes that that encode proteins that help the organism encode proteins that help the organism survive heat stress. These genes are survive heat stress. These genes are activated under conditions of heat activated under conditions of heat stress, under the control of a stress, under the control of a transcription factor called transcription factor called heat shock heat shock transcription factortranscription factor. This factor is . This factor is always present, but is only activated always present, but is only activated when greatly increased temperatures when greatly increased temperatures are detected. are detected.

• Whereas enhancers help to increase Whereas enhancers help to increase the transcriptional activity of genes, the transcriptional activity of genes, other DNA sequences, known as other DNA sequences, known as silencers help to repress the silencers help to repress the transcription of genes through a transcription of genes through a similar series of interactions.similar series of interactions.