Regulation of Gene Expression

Chapter 18 Regulation of Gene Expression

Gene expression: Bacteria vs. Eukaryotes • prokaryotes and eukaryotes alter gene expression in response to their changing environment • gene expression = refers to the entire process whereby genetic information is decoded into a protein • prokaryotes and eukaryotes carry out gene expression in similar ways • transcription using an RNA polymerase • translation using ribosomes • but there are some differences: • 1. RNA polymerases differ – only one in prokaryotes; 3 in eukaryotes • 2. transcription factors used by eukaryotes • 3. transcription is terminated differently in prokaryotes vs. eukaryotes • 4. ribosomes – bacterial ones are smaller • 5. lack of compartmentalization in bacteria – transcribe and translate at the same time

So what is a gene? unit of inheritance located on chromosomes region of specific nucleotide sequence located along the length of DNA DNA sequence that codes for a specific sequence of amino acids BUT: some DNA sequences are NEVER translated e.g. rRNA and tRNA are transcribed but not translated into anything so a gene is a region of DNA that is either 1. translated into a sequence of amino acids (polypeptide)  functional protein 2. transcribed into a RNA molecule

So what is a gene? molecular components of a gene: A. coding sequences - eukaryotes have introns within their coding sequence B. promoter C. enhancers – found in eukaryotes D. UTRs – found in eukaryotes E. poly-adenylation sequence – found within the eukaryotic 3’ UTR

Overview: Conducting the Genetic Orchestra genetic and biochemical work in bacteria identified two things 1. protein-binding regulatory sequences associated with genes 2. proteins that can bind these regulatory sequences – either activating or repressing gene expression these two components underlie the ability of both prokaryotic and eukaryotic cells to turn genes on and off

Precursor Feedbackinhibition Bacteria often respond to environmental change by regulating transcription trpE gene Enzyme 1 trpD gene • natural selection has favored bacteria that produce only the products needed by that cell • bacteria regulate the production of enzymes by feedback inhibition or by gene regulation • gene expression in bacteria is controlled by the operon model Regulationof geneexpression Enzyme 2 trpC gene  trpB gene  Enzyme 3 trpA gene Tryptophan (b) (a) Regulation of enzymeactivity Regulation of enzymeproduction

Operons: Definitions & Concepts • bacteria group functionally related genes so they can be under coordinated controlby a single “on-off regulatory switch” • the regulatory “switch” is a segment of DNA called an operator • binding sites for transcription factors that help RNA polymerase II bind the nearby promoter • the operator can be controlled by proteins or nutrients • e.g. can be switched off by a protein called a repressor • repressor prevents gene transcription - binds to the operator and blocks RNA polymerase binding to the promoter • repressor is the product of a separate regulatory gene • repressor can be in an active or inactive form, depending on the presence of other molecules • co-repressor is a molecule that cooperates with a repressor protein to switch an operon off • e.g. the amino acid tryptophan

Operons: Definitions & Concepts • operon = the entire stretch of DNA that includes the operator, the promoter, and the genes that the promoter controls • the transcription of the downstream genes is polycistronic • produces one long piece of mRNA containing multiple transcription units • two kinds – “On” and “Off” operons trp operon Promoter Genes of operon trpE trpD trpC trpA trpB Operator Start codon Stop codon mRNA 5 E D C B A Polypeptide subunits that make upenzymes for tryptophan synthesis

Repressible and Inducible Operons: Two Types of Negative Gene Regulation • OFF operon = repressible operon is one that is usually on but is turned OFF by a repressor • e.g. the trp operon is a repressible operon • ON operon = inducible operon is one that is usually off but is turned ON by an inducer • e.g.lac operon is an inducible operon

The trp Operon: Repressible Operons • E. coli can synthesize the amino acid tryptophan when it is absent from the growth media • by default the trp operon is on and the genes for tryptophan synthesis are transcribed • comprised of the: • 1. operator – capable of binding a repressor protein • 2. genes of the operon – for synthesizing tryptophan when it is missing from the growth media • plus a regulatory gene = trpR • expressed whether tryptophan is absent or present

The trp Operon: Repressible Operons trp operon Promoter Promoter Genes of operon DNA trpE trpD trpC trpA trpR trpB Operator Regulatory gene RNApolymerase Start codon Stop codon 3 mRNA 5 mRNA 5 E D C B A Protein Inactive repressor Polypeptide subunits that make upenzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on • when tryptophan is absent – the operon needs to function to make tryptophan • the repressor protein is made but it is inactive & is incapable of binding the operator • RNA polymerase can bind the promoter and the downstream genes are expressed

The trp Operon: Repressible Operons DNA No RNAmade • when tryptophan is present – the operon does not need to be functional • tryptophan acts as a co-repressor & binds the repressor protein • this allows the repressor to bind and repress the function of the operator • MUCH lower downstream gene expression vs. when the operon is ON mRNA Protein Activerepressor Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off

The lac Operon: Inducible Operons • proposed by Francois Jacob and Jacques Monod - 1960s • E.coli can use glucose and other sugars (such as lactose) as their sole source of carbon and energy • the normal situation is for the bacteria to use glucose • levels of a bacterial enzyme called beta-galactosidase (lactose breakdown) are very low • when lactose is given to the bacteria – b-Gal levels increase • said to be induced • the lac operon is an inducible operon • contains genes that code for enzymes used in the hydrolysis and metabolism of lactose • when E. coli are grown with glucose – no need for the enzymes of the lac operon since there is no lactose in the medium • so the operon is turned OFF • but with media containing lactose – need to turn the operon ON to make the enzymes for metabolizing and using lactose

The lac Operon: Inducible Operons • genes of the lac-operon: • 1. lacZ gene = beta-galactosidase – splits the lactose into glucose and galactose • 2. lacY gene • 3. lacA gene • 4. lacI gene = codes for a lac repressor • 5. operator = binds transcription factors • 6. promoter = binds RNA polymerase II • THIS IS IMPORTANT!!! - without any outside control - the lac repressor gene lacI is constitutively active and acts to eventually switch the lac operon OFF • through the constitutive production of a lac repressor protein • a molecule called an inducer is needed to inactivate the repressor to turn the lac operon ON

Regulatorygene Promoter Operator lacI lacZ DNA DNA NoRNAmade 3 mRNA RNApolymerase 5 Activerepressor Protein (a) Lactose absent, repressor active, operon off lac repressor protein • when lactose is absent – an active repressor is made • the genes metabolizing lactose are NOT needed • repressor gene lacI is constitutively active – makes a lactose repressor • repressor binds the operator and hinders the binding of the RNA polymerase to the promoter • downstream genes are transcribed AT A VERY LOW LEVEL

when lactose is present – an inducer is required to turn the operon ON • metabolizing and using lactose is now needed • ALLOLACTOSE ACTS AS AN INDUCER • allolactose – form of lactose that can enter bacterial cells • the inducer binds the repressor and prevents it from binding to the operator • the downstream genes are expressed AT A HIGH LEVEL • lactose binding to the repressor shifts the repressor to its non-DNA binding conformation lac operon lacI lacZ lacY lacA DNA RNA polymerase 3 mRNA mRNA 5 5 -Galactosidase Permease Transacetylase Protein Inactiverepressor Allolactose(inducer) (b) Lactose present, repressor inactive, operon on

in nature – the inducer of the lab operon is a lactose derivative • in the lab – other inducers can be used to turn the operon on • e.g. IPTG = isopropyl-b-D-thiogalactoside • IPTG is NOT a substrate of b-Gal • we can also give the bacteria a specific b-Gal substrate that will turn colors • X-Gal – turns blue with broken down by b-gal enzyme • used to identify bacteria containing cloned genes • can insert additional genes into plasmids containing the b-gal gene • insertion of your desired gene INTO the plasmid disrupts b-gal expression • inability to breakdown X-Gal – colonies are white

inducible enzymes usually function in catabolic pathways their synthesis is induced by a chemical signal repressible enzymes usually function in anabolic pathways their synthesis is repressed by high levels of the end product regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor

Positive Gene Regulation: CAP proteins • some operons are also subject to positive control • when bacteria are given both lactose AND glucose - the bacteria will use glucose • the enzymes for glycolysis are continually present in bacteria • when lactose is present and glucose is short supply – it makes the enzymes for lactose metabolism • how does the bacteria sense the low levels of glucose??

Promoter DNA lacI lacZ Operator CAP-binding site RNApolymerasebinds andtranscribes ActiveCAP cAMP Inactive lacrepressor InactiveCAP Allolactose (a) Lactose present, glucose scarce (cAMP level high):abundant lac mRNA synthesized -when glucose is scarce  accumulation of a small molecule called cyclic AMP (cAMP) -cAMP functions as a “2nd messanger”to signal that glucose levels are low in the growth medium - high levels of cAMP activate a regulatory protein called catabolite activator protein (CAP) -cAMP binds CAP and activates it - activated CAP attaches to the lac operon promoter and accelerates transcription (functions as a transcription factor) - enhances the affinity of RNA polymerase for the promoter

CAP helps regulate other operons that encode enzymes used in catabolic pathways • when glucose levels are low and lactose levels are high • 1. lactose binds the lactose repressor and prevents it from binding the operator and inhibiting gene transcription = genes for lactose metabolism are made • 2. cAMP activation of CAP and its binding to the lac promoter increases transcription = lactose genes are made at a higher rate

when glucose levels increase and lactose levels decrease • 1. CAP activation will eventually decrease and so will its enhancement of transcription • 2. the lactose repressor is now able to bind the operator and inhibit transcription

so the lac operon is actually under dual control as lactose increases and glucose decreases: • positive – as levels of cAMP rise – so does CAP activation and the activity of the lac operon • negative – as repressor activity decreases & the activity of the lac operon increases • THEREFORE: it is the allosteric state of the lac repressor that determines if transcription happens • it is the presence of CAP that controls the rate at which transcription will happen

Signal NUCLEUS Eukaryotic gene expression is regulated at many stages Chromatin Chromatin modification:DNA unpacking involvinghistone acetylation andDNA demethylation DNA Gene availablefor transcription Gene Transcription • all organisms must regulate which genes are expressed at any given time • in the same organism – the genomes are identical from cell to cell • so why do different cells express different genes/proteins?? • differences result from differential gene expression = the expression of different genes by cells with the same genome • several steps along the replication/transcription/translation path are control points for differential gene expression • control of DNA transcription – modification of DNA-histone interaction • post-transcriptional control • post-translational control Exon RNA Primary transcript Intron RNA processing Tail mRNA in nucleus Cap Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Translation Degradationof mRNA Polypeptide Protein processing, suchas cleavage and chemical modification Active protein Degradationof protein Transport to cellulardestination Cellular function (suchas enzymatic activity,structural support)

each of the histone proteins (H2A, H2B, H3, H4) contain flexible extensions of 20 to 40 amino acids called “tails” these histones can be modified post-translationally by the addition of functional groups at the end of these tails are several positively charged lysine amino acids some of these lysines undergo reversible chemical modification called acetylation important for transcription, resistance against DNA degradation Histones Histones Control of DNA Transcription: Histone Acetylation

numerous post-translational modifications can be done to histone proteins affects how the DNA-histone interacts and ultimately affects the transcription of the DNA some histone lysines undergo reversible chemical modifications called acetylation and deacteylation acetylation = transfer of an acetyl group onto the NH2 terminus of an amino acid for histones – performed by a family of enzymes called histone acteyltransferases (HATs) acetylation neutralizes the +ve charge of these lysines its interaction with the DNA is eliminated the DNA becomes less tightly associated with the histone results in better access for the transcriptional machinery to the DNA acetyl coA “donor” lysine R-group Acetylation and Deacetylation of DNA

deacetylation = removal of this acetyl group from the histone by a family of enzymes called histone deacetylases (HDACs) increases the interaction between DNA and the histone by removing the acetyl group and increasing the “positivity” of the lysine residues 11 eukaryotic HDACs !!! acetyl coA “donor” lysine R-group Acetylation and Deacetylation of DNA

acetylation/deacetylation is a transient histone modification that affects transcription euchromatin – higher HAT activity  more transcriptionally active form of chromatin heterochromatin – higher HDAC activity  less transcriptionally active form of chromatin heterochromatin Increased binding of transcription factors and RNA Pol II to “opened” acetylated chromatin Protein euchromatin Control of DNA Transcription: Acetylation and Deactylation of DNA

the HAT/HDAC enzymes are part of a large complex of proteins that binds the DNA includes transcription factors, other regulatory proteins, RNA polymerase II it is now thought that HAT and HDAC enzymes are recruited into this complex once there – they modify the DNA and give the rest of the transcription machine better “access” to the DNA helix non-histone proteins can also be acetylated!! e.g. transcription factors are also acetylated/deacetylated – changes their activity level and therefore transcription Control of DNA Transcription: Acetylation and Deacetylation of DNA

Histone Methylation • histone methylation= the addition of methyl groups (CH3) to certain amino acids on histone tails • lysines or arginines – usually lysines • is associated with reduced transcription in cases, increased transcription in others • usually results in increased association between the histone and the DNA and a decrease in transcription in that area • histone methylation is considered an epigenetic modification • alteration of gene expression by mechanisms outside of DNA structure • performed by a family of enzymes called histone methyltransferases

DNA Methylation • in addition to histones – methyl groups can be attached to certain DNA bases = DNA methylation • usually cytosine • done by a different set of enzymes than those that methylate histones • is associated with reduced transcription in some species • i.e. the more methylated, the more inactive the gene • DNA methylation essential for long-term inactivation of genes during cellular differentiation • DNA methylation can last through several rounds of replication • when a methylated DNA sequence is replicated – the daughter strand is methylated too • can affect transcription rates over several rounds of replication

Regulation of Transcription Initiation • chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machinery • additional transcriptional levels are also found • enhancers • promoters

Organization of a Typical Eukaryotic Gene Enhancer(distal controlelements) Poly-Asignalsequence Proximalcontrolelements Transcriptionterminationregion Transcriptionstart site • most eukaryotic genes are associated with multiple control elements • segments of noncoding DNA that serve as binding sites for transcription factors that help regulate transcription • distal elements– known as enhancers • proximal elements – associated with promoters • many of these control elements and the transcription factors they bind are responsible for the differential gene expression seen in different cell types Exon Intron Intron Exon Exon DNA Upstream Downstream Promoter

Transcription Factors • proteins that bind sequences of DNA to control transcription • can act as activators or repressors to transcription • activating TFs - proteins that recruit the RNA polymerase to a promoter region • repressing TFs – proteins that prevent transcription in many ways • must contain a DNA binding domain to be a transcription factor • not always one protein – can be multiple subunits together in a complex • two broad categories: • 1. general transcription factors • 2. specific transcription factors

Transcription Factors • two broad categories: • 1. general transcription factors are essential for the transcription of all protein-coding genes • assist the RNA polymerase in binding the promoter region – only give a low level of transcription!! • activity is enhanced by specific transcription factors • 2. specific transcription factors control the high-level, differential expression of specific genes within a specific cell type • bind the promoter and enhancer regions of a gene • can function to activate or repress transcription • e.g. Runx-2 – transcription factor that is found in osteoblasts • directs the expression of several osteogenic genes involved in making bone

Transcription Factors bind DNA • binding of a TF to DNA is easy to see experimentally using a DNA mobility shift assay • Step #1 – radioactively label your DNA • Step #2 – mix your labelled DNA with your possible TFs • Step #3 – Southern blot - run the DNA on a gel and transfer it to a filter paper -control – DNA not mixed with the possible TF • Step #4 – expose the filter to film to develop the radioactive signal DNA footprinting assay

Promoters • sequence of DNA located immediately upstream of the transcription start site • promotes transcription of DNA into RNA • site of RNA polymerase binding in both prokaryotes and eukaryotes • contain sequences for the binding of RNA polymerase and sequences for the binding of transcription factors

Promoters • initial work done in bacteria • found two kinds of DNA sequences in the promoter • 1. those that are found in the promoters of all bacterial genes • 2. those that are found in a more limited number of genes that respond to a specific signal • core bacterial promoter: binds RNA polymerase and an associated sigma factor (part of the RNA polymerase complex) • two key DNA sequences located 10 and 35 bp upstream from the transcription start site (TSS) – for the binding of the RNA polymerase • -10 site = TATA box (consensus sequence TATAAT) • -35 site - TTGACA

Promoters • eukaryotic promoters: 10 classes of promoters known • DEFINITION: DNA sequence that binds the RNA polymerase II (plus 7 additional factors) • BUT – more complicated than prokaryotic promoters • are additional upstream DNA sequences that regulate transcription • three well-known regions studied • -30, -75 and -90 sites • two kinds of DNA sequences associated with transcription of a eukaryotic gene: • 1. part of the promoter (i.e. control elements) involved in the basic process of transcription • 2. control elements active in a particular tissue type or in response to a specific signal – regulated transcription

Promoters • promoter sequences involved in basic transcription • TATA box – conserved from the bacterial TATA box • A/T rich sequence - 30 base pairs upstream of start site • in most genes but not all – not found in housekeeping genes • together with the transcription start site – considered to be the core promoter • accurately positions the RNA polymerase at the start site • also binds general transcription factors • actually provides a very low level of transcription • needs upstream promoter elements (UPEs) to increase transcriptional control • UPEs – also essential for transcription • increase the efficiency of transcription • e.g. SP1 sequence &/or CCAAT box • some genes can have an SP1 and CCAAT box – e.g. hsp70 gene • others may only have one – e.g. metallothionein gene

promoters sequences involved in regulated transcription • promoters also have control elements that are shared with a more limited number of genes • interspersed among the UPEs • provide cell-type specific or signal-specific transcription • some well-known sequences are known as Response Elements • e.g. metallothionein gene – metal response elements (MREs) • gene binds metals such as zinc – acts to decrease oxidative stress • many hormones act through these response elements • e.g. estrogen Histone 2A gene promoter Metallothionein 1 gene promoter

Promoters • functions of promoter were discovered through • mutation or deletion of specific regions • putting these regions in front of a reporter gene  is the reporter gene transcribed? • e.g. insertion of the metallothionein gene promoter into a reporter plasmid containing the firefly luciferase gene (reporter gene) • transcription of the luciferase gene will be driven by the inserted promoter • put in the entire promoter and measure luciferase activity • put in “pieces” of the promoter with specific regions deleted – measure luciferase activity Activation of Metallothionein Gene Expression by Hypoxia Involves Metal Response Elements and Metal Transcription Factor-1 Cancer Res March 15, 1999 vol. 59 no. 6 pp1315-1322

Enhancers Enhancer(distal controlelements) Poly-Asignalsequence Proximalcontrolelements Transcriptionterminationregion Transcriptionstart site • distal control elements of a gene • DNA sequences that act to enhance eukaryotic transcription • can be found either: • upstream of the gene • downstream of the gene • within the gene • even on a different chromosome!!! • act to increase the activity of the promoter • DO NOT have promoter activity themselves • some enhancers are active in all tissues and increase promoter activity constitutively • others are only expressed in specific cells • made up of several DNA sequences (sequence elements) that bind transcription factors which interact together • many of these sequence elements are also found in the promoter Exon Intron Intron Exon Exon DNA Upstream Downstream Promoter

Enhancers & their Transcription Factors • transcription factors than bind enhancers are called activators • positively acting transcription factors • activators= proteins that bind to DNA sequences and stimulate/activate transcription of a gene • activators have two domains • 1. DNA binding domain • 2. activation domain - site that activates transcription by helping to form the transcription initiation complex Activationdomain DNA-bindingdomain DNA

Eukaryotic gene elements: a summary • so the typical eukaryotic gene consists of up to 4 distinct control elements • 1. core promoter itself – upstream of the transcription start site • 2. upstream promoter elements (UPEs) located close to the promoter – required for efficient transcription in any cell • 3. elements that intersperse among the UPEs and activate transcription of genes in specific tissues or in response to specific stimuli – regulated transcription elements • 4. distant elements called enhancers

Transcription Initiation • transcription can happen as long as the core promoter is present • but transcription rates will be very low • so efficient transcription of eukaryotic genes requires the activity of the promoter, enhancers and a multitude of transcription factors together with the RNA polymerase II • these components come together to form atranscription initiation complex • stepwise assembly • 1. binding of three general transcription factors at the TATA box– TFIIA, TFIIDTFIIB • 2. recruitment and binding of the RNA polymerase II at the TF/TATA box complex • in some organisms – polymerase is bound to these 3 TFs before binding DNA first = RNA polymerase holoenzyme • 3. additional general TFs join • 4. binding of gene-specific TFs + interaction with enhancer/activators

Promoter Activators Gene DNA Distal controlelement TATA box -activators bind to the DNA of the enhancer via their DNA-binding domains -the activators bind to regions called distal control elements Enhancer Generaltranscriptionfactors DNA-bendingprotein a DNA bending protein “bends” the distal enhancer region – bringing it close to the the promoter Group of mediator proteins RNApolymerase II general transcription factors, promoter-specific TFs, mediator proteins and RNA polymerase II form a transcription initiation complex with the enhancer and its activators RNApolymerase II Transcriptioninitiation complex RNA synthesis

Transcription Initiation promoter-specific transcription factors activator-enhancer Mediator protein = co-activator -multi-subunit protein complex found in the transcription initiation complex of all eukaryotes -31 proteins!!!! -functions as a bridge between transcription factors

Repressors • some transcription factors can also function as repressors or silencers • inhibiting expression of a particular gene by a variety of methods • some repressors bind activators and prevent their binding to enhancers • some bind the distal control elements in the enhancer directly • others bind proximal control elements or the promoter

Cell-Type Specific Transcription Enhancer Promoter LIVER CELLNUCLEUS Controlelements Albumin gene Availableactivators Crystallingene • both liver and lens cells have the same genome • so why does a liver cell make albumin and a lens cell make crystallin????? • it’s the transcription factors and control elements • liver cell has a unique complement of transcription factors that activate albumin transcription Albumin geneexpressed Crystallin genenot expressed (a) Liver cell

Regulation of Gene Expression