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LECTURE PRESENTATIONS For CAMPBELL BIOLOGY, NINTH EDITION Jane B. Reece, Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, Robert B. Jackson Chapter 18 Regulation of Gene Expression Lectures by Erin Barley Kathleen Fitzpatrick © 2011 Pearson Education, Inc. 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 • eukaryotes – polyA signal is transcribed prior to termination – 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 – it is only the mRNA that is translated • 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 – only found in eukaryotes – D. UTRs – only 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 • known as promoter and enhancer regions – 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 Bacteria often respond to environmental change by regulating transcription • 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 • in bacteria – half of the genes are clustered into operons Precursor Feedback inhibition trpE gene Enzyme 1 trpD gene Enzyme 2 trpC gene trpB gene Enzyme 3 – genes encode the enzymes for a metabolic pathway • all the genes are transcribed into ONE mRNA Regulation of gene expression trpA gene Tryptophan (a) Regulation of enzyme activity (b) Regulation of enzyme production Operons: Definitions & Concepts • bacteria group functionally related genes so they can be under coordinated control by a single “on-off regulatory switch” • the regulatory “switch” is a segment of DNA called an operator – usually positioned downstream of the promoter – 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 • transcription produces a long piece of mRNA containing multiple transcription units • two kinds – “On” and “Off” operons trp operon Promoter trpE Operator Start codon mRNA 5 Genes of operon trpD trpC trpB trpA B A Stop codon E D C Polypeptide subunits that make up enzymes 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 trpR Regulatory gene mRNA 3 RNA polymerase Operator Start codon trpD trpC trpB trpA C B A Stop codon mRNA 5 5 E Protein Inactive repressor (a) Tryptophan absent, repressor inactive, operon on D Polypeptide subunits that make up enzymes for tryptophan synthesis •when tryptophan is absent – the operon needs to function to make tryptophan •the repressor protein is made but it is inactive •inactive repressor is incapable of binding the operator •RNA polymerase can bind the promoter and the downstream genes are expressed AT A HIGH LEVEL The trp Operon: Repressible Operons DNA No RNA made mRNA Protein Active repressor Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off -the binding of tryptophan changes the shape of the active site of the repressor -allosteric regulation: regulation of an enzyme (or a protein) via the binding of a molecule to a site other than the active site •when tryptophan is present – the operon does not need to be functional •tryptophan acts as the co-repressor •it binds the repressor protein •changes the shape of the repressor and activates it - allows the repressor to bind and repress the function of the operator •MUCH LOWER downstream gene expression http://www.youtube.com/watch?v=8aAYtMa3GFU http://bcs.whfreeman.com/thelifewire/content/chp13/1302002.html 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 a 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 • can also convert lactose into allolactose – – – – – 2. lacY gene = lactose permease – pumps lactose into the bacterial cell 3. lacA gene = thiogalactase transacetylase – function??? 4. lacI gene = codes for a lac repressor 5. operator = binds transcription factors 6. promoter = binds RNA polymerase • 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 Regulatory gene Promoter Operator lacZ lacI DNA No RNA made 3 mRNA 5 Protein RNA polymerase Active repressor (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 •the 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 the enters 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 concentration of the repressor to its non-DNA binding conformation lac operon lacI DNA lacZ lacY b-Galactosidase Permease lacA RNA polymerase 3 mRNA 5 mRNA 5 Protein Allolactose (inducer) Inactive repressor (b) Lactose present, repressor inactive, operon on Transacetylase -the mechanism is shut off upon metabolism of the allolactose -b-gal can hydrolyze allolactose • in nature – the inducer of the lab operon is a lactose derivative called allolactose • in the lab – other inducers can be used to turn the operon on – e.g. IPTG = isopropyl-b-D-thiogalactoside (allolactose mimicker) – IPTG is NOT metabolized by b-Gal – its concentration will remain constant in the cell and so will its induction of b-gal expression • 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 CAP-binding site cAMP Operator RNA polymerase Active binds and transcribes CAP Inactive CAP Allolactose Inactive lac repressor (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 GO HOME AND STUDY Next Lecture EUKARYOTIC GENE REGULATION Eukaryotic gene expression is regulated at many stages Signal NUCLEUS Chromatin • • • • • 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 DNA Chromatin modification: DNA unpacking involving histone acetylation and DNA demethylation Gene available for transcription Gene Transcription RNA Cap Exon Primary transcript Intron RNA processing Tail mRNA in nucleus Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Polypeptide Protein processing, such as cleavage and chemical modification Degradation of protein Active protein Transport to cellular destination Cellular function (such as enzymatic activity, structural support) Control of DNA Transcription: Histone Acetylation • 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 posttranslationally by the addition of chemical • at the end of these tails are several positively charged lysine amino acids – some interact with the negatively charged DNA in their nucleosome – others interact with the linker DNA in between histones – others interact with neighboring nucleosomes • some lysines undergo reversible chemical modification called acetylation – important for transcription, resistance against DNA degradation Histones Histones Acetylation and Deacetylation of DNA • numerous chemical modifications can be done to histone proteins – e.g. methylation, phosphorylation – affects how the DNA-histone interacts and ultimately affects the transcription of the DNA • some histone lysines undergo reversible chemical modifications called acetylation and deacetylation • acetylation = transfer of an acetyl group onto the NH2 terminus of an amino acid – for histones – performed by a family of enzymes called histone acetyltransferases (HATs) – uses acetyl coA as the source of the acetyl group – principle targets are the H3 and H4 histone subunits • 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 (e.g. RNA pol II) to the DNA acetyl higher rates of transcription result 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 contact 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 Control of DNA Transcription: Acetylation and Deactylation 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 euchromatin Increased binding of transcription factors and RNA Pol II to “opened” acetylated chromatin Protein Control of DNA Transcription: Acetylation and Deacetylation 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 by transcription factors – 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. acetylation of wood proteins increases their hardness 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 -depends on the amino acid methylated and the number of methyl groups added • e. g. one methyl group added- increased chromatin condensation • two methyl groups added – decreased chromatin condensation – 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 • the inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance 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 control elements) DNA Upstream Proximal control elements Transcription start site Exon Promoter Intron Exon Intron Poly-A signal sequence Exon Transcription termination region Downstream • 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 – known as enhancers – proximal – associated with promoters • these control elements and the transcription factors they bind are responsible for the differential gene expression seen in different cell types Transcription Factors • proteins that bind sequences of DNA to control transcription in eukaryotes • 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 • activators have two domains Activation domain DNA-binding domain DNA 1. DNA binding domain 2. activation domain - site that activates transcription by helping to form the transcription initiation complex 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 Promoters • • • • sequences 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 in eukaryotes the promoter also binds transcription factors Promoters • 1. Core promoter – found in most eukaryotic genes – gives a low level of transcription • 2. proximal promoter elements/promoter proximal elements (PPEs) – found in addition to the core in many genes – gives regulated transcription – e.g. in certain cells at certain times Core promoter • in bacteria: binds RNA polymerase and an associated sigma factor (part of the RNA polymerase complex) – 10 nucleotides upstream from the start site of transcription is a key region = TATA box (consensus sequence TATAAT) – is another site – called the -35 site – TTGACA – the core promoter binds a sigma factor called s70 – recognizes the TATA box Core Promoter • in eukaryotes: – TATA box – conserved from the bacterial TATA box • • • • A/T rich sequence - ~ 30 base pairs upstream of start site (-30 position) in most genes but not all – not found in housekeeping genes together with the transcription start site – considered to be the core promoter like the bacterial promoter – TATA box accurately positions the RNA polymerase at the start site • it also binds general transcription factors • BUT needs proximal promoter elements (PPEs) to increase transcriptional control The Core Promoter binds General Transcription Factors • the core promoter binds general TFs • there is a specific order to binding – 1. binding of first three general transcription factors at the TATA box– TFIIA, TFIID & TFIIB – 2. binding of the RNA polymerase II at the TF/TATA box complex – 3. additional general TFs join – 4. binding of gene-specific TFs + interaction with activators bound to the enhancer Additional Control Elements for Transcription • core promoter only provides basic control over transcription • eukaryotic genes have additional control elements to better control • 1. proximal promoter elements/promoter proximal elements (PPEs) • 2. enhancer sequence elements or enhancers Proximal Promoter Elements (PPEs) • located further upstream (or downstream) of the core promoter – couple of hundred base pairs upstream • increase the efficiency of transcription by binding additional transcription factors • numerous types – most common: • 1. octamer (Oct) consensus • 2. GC-rich regions • 3. CCAAT box most eukaryotic genes use a combination Proximal Promoter Elements (PPEs) Histone 2A gene promoter Proximal Promoter Elements (PPEs) • PPEs are bound by cell-type specific transcription factors • work with the TATA box (with its general transcription factors and RNA polymerase) in the initiation of transcription Enhancers • distal control elements of a gene • are DNA sequences called sequence elements that act to enhance eukaryotic transcription • bind transcription factors called activators • the enhancer interacts with the promoter to enhance transcription Enhancer (distal control elements) DNA Upstream Proximal control elements Transcription start site Exon Promoter Intron Exon Intron Poly-A signal sequence Exon Transcription termination region Downstream Enhancers Eukaryotic gene elements: a summary • so the typical eukaryotic gene consists of up to 3 distinct control elements – 1. core promoter– upstream of the transcription start site • for basic transcriptional control – 2. promoter proximal elements (PPEs) located close to the promoter • required for efficient transcription in any cell – increase transcription rate – 3. 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 core promoter, PPEs, enhancers and a multitude of transcription factors together with the RNA polymerase II • these components come together to form a transcription initiation complex Transcription Initiation Promoter Activators Gene DNA TATA box Enhancer • General transcription factors activators bind to the distal enhancer DNAbending protein • a DNA bending protein “bends” the distal part of the DNA – bringing it close to the promoter Group of mediator proteins RNA polymerase II • • general TFs, PPE-specific transcription factors, mediator proteins and RNA polymerase II are added form a transcription initiation complex with the enhancer and its activator/transcription factors RNA polymerase II Transcription initiation complex RNA synthesis Transcription Initiation – Pretty Picture, eh? activator-enhancer promoter-specific 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 Control elements LIVER CELL NUCLEUS Albumin gene Crystallin gene Available activators specific for liver genes • 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 gene expressed Crystallin gene not expressed (a) Liver cell Enhancer Control elements Promoter LENS CELL NUCLEUS Albumin gene Crystallin gene • the lens cell has a different set of TFs that activates transcription in these cells • these transcription factors may only be made within a lens or liver cell at a precise time in development or in response to an extracellular signal (e.g. growth factor or hormone) or even an environment cue Available activators specific for lens genes Albumin gene not expressed Crystallin gene expressed (b) Lens cell Go have lunch! Next lecture: Post-transcriptional and translational control Mechanisms of Post-Transcriptional Regulation • regulatory mechanisms can operate at various stages after transcription • allow a cell to fine-tune gene expression rapidly in response to environmental changes • post-transcriptional processing: – 1. mRNA structure – cap and tail; UTRs – 2. mRNA splicing – 3. mRNA half life and degradation Post-Transcriptional Regulation: mRNA structure • pre-RNA processing to mRNA involves the addition of the 5’ methylated cap and 3’ poly-A tail • cap is added shortly after transcription initiation – by a capping enzyme which is associated with the RNA polymerase II – cap - 7-methylguanosine Post-Transcriptional Regulation: mRNA structure – function of the cap • 1. protection of mRNA against degradation • 2. export of mRNA out into the cytoplasm • 3. binding of the small subunit to mRNA for translation Post-Transcriptional Regulation: mRNA structure • cap is followed by the 5’UTR region (untranslated region) – part of the transcription unit – found between the transcription start site and ends one nucleotide before the ATG/start codon of the coding sequence – contains sequences for controlling gene expression, mRNA export, translation initiation – in bacteria – contains a sequence for docking of the ribosome = Shine Delgarno Sequence TSS Post-Transcriptional Regulation: mRNA structure • poly A tail – in animal cells, all mRNAs (except histone mRNAs) have polyA tails – prevent degradation of the mRNA and induces export from the nucleus – “A” nucleotides are added following enzymatic cleavage of the mRNA Post-Transcriptional Regulation: mRNA structure • poly A tail – two special mRNA sequences are needed – located in the 3’UTR • 1. Poly A signal – AAUAAA • 2. Poly A site – downstream from the signal – area rich in Gs ands Us -a Poly(A) polymerase binds the poly A signal – cleaves the mRNA at the poly A site and adds hundreds of “A” nucleotides Post-Transcriptional Regulation: mRNA structure • 3’UTR – second of the two UTRs that flank a transcription unit’s coding sequence – much longer than the 5’UTR – average is 800 NTs long – length is important – longer the UTR, the lower level of gene expression (due to miRNA action) – numerous regions for a variety of functions Post-Transcriptional Regulation: mRNA structure • 3’UTR - contains numerous regulatory regions for • 1. poly–adenylation – contains the polyA signal and polyA site • 2. mRNA stability – contain AU-rich elements (AREs) that increase the stability of the mRNA; also interacts with miRNAs (repress translation and degrade mRNA) • 3. mRNA export – contains sequences that attract nuclear export proteins + attract proteins that will associate the mRNA with the cytoskeleton • 4. improves translation efficiency Post-transcriptional control: mRNA degradation • • • • the life span of mRNA molecules in the cytoplasm is a key to determining protein synthesis eukaryotic mRNA is more long lived than prokaryotic mRNA numerous enzymes (RNases) can breakdown mRNA the binding of small RNAs called microRNAs (miRNAs) to the mRNA can target it for degradation Post-transcriptional Regulation: Splicing • removal of introns and the joining of exons – for short mRNAs – happens after poly-adenylation at the 3’ end – for longer mRNAs with multiple exons – happens during transcription • performed in the nucleus by the spliceosome – small nuclear RNAs – U1, U2, U4, U5 and U6 – associated with protein subunits = snRNPs – snRNPs form the core of the spliceosome Post-transcriptional Regulation: Splicing • spliceosome recognizes conserved sequences at the start and end of an intron = splice sites • introns are removed as a lariat structure – a 5’ G at the start of the intron is joined in an unusual phosphodiester bond (2’ to 5’) to the A at the end of the intron – “A” nucleotide is called a branch point Post-transcriptional Regulation: Splicing • the snRNPs are numbered based on their “entrance” into the splicing reaction • 1. first is U1 – base pairs with the 5’ G at the start of the intron • 2. next is U2 which binds the branch point A • 3. U4/U6 and U5 enter next – formation of the completed spliceosome • 4. rearrangements among these snRNAs “loops out” the intron and cuts the intron at the 5’ end • 5. departure of U1 and U4 + joining of the 5’ end of the intron to the A (completes the lariat) • 6. U6 snRNA cuts at the 3’ end of the intron and joins the two exon sequences Differential Splicing • In differential RNA splicing, different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns Exons DNA 1 4 3 2 5 Troponin T gene Primary RNA transcript 3 2 1 5 4 RNA splicing mRNA 1 2 3 5 or 1 2 4 5 Protein Processing • after translation - various types of protein processing, including folding, cleavage and the addition of chemical groups take place • known as post-translational processing • numerous kinds of chemical additions Post-translational modifications of Proteins • numerous chemical groups can be added – – – – – a. phosphorylation b. methylation c. acetylation d. glycosylation e. iodination Protein Folding: Chaperones • protein function is completely dependent upon 3D structure • the information for folding is contained within the amino acid sequence of the polypeptide chain • • • – the hydrophobic residues are “buried” within the center of the folding protein – spontaneous process – large numbers of interactions between the R groups of the AAs form • ionic • van der waals – between hydrophobic groups • disulfide bridges folding can begin the minute the PP chain emerges from the ribosome but most proteins don’t fold the quickly these proteins are met at the ribosome by a class of proteins called molecular chaperones Protein Folding: Chaperones • molecular chaperones – best described class of chaperones – heat shock proteins or HSPs – work by interacting with exposed hydrophobic AAs – hydrophobic residues are dangerous – e.g. HSP60 forms a “barrel-like” structure that “isolates” folding proteins AFTER they are made = known as chaperonin – bind to the hydrophobic residues and ensures correct folding Proteasome and ubiquitin to be recycled Ubiquitin Proteasome Protein to be degraded Ubiquitinated protein Protein entering a proteasome Protein fragments (peptides) • if the protein is not folded properly – will have to be degraded • Proteasomes are giant protein complexes that bind protein molecules and degrade them • consists of a protein complex that forms a hollow cylinder (20S core) • top and bottom are additional protein complexes that feed the abnormal protein into the core (19S cap) – protein is unfolded as it is fed in – exposed to proteases within the core – keeps the protein in the core until the entire protein is cleaved into peptides Proteasome and ubiquitin to be recycled Ubiquitin Proteasome Protein to be degraded Ubiquitinated protein Protein entering a proteasome • signal to enter the proteosome is the chemical attachment of a poly-ubiquitin chain Protein fragments (peptides) Noncoding RNAs play multiple roles in controlling gene expression • only a small fraction of DNA codes for proteins – 30,000 genes • a fraction of the non-protein-coding DNA pieces are genes for RNAs such as rRNA and tRNA • but most are transcribed into noncoding RNAs (ncRNAs) – e.g. miRNA – e.g. siRNA – e.g. snRNA MicroRNAs • MicroRNAs (miRNAs) are small singlestranded RNA molecules that can bind to mRNA • described between 2000 and 2005 • can degrade mRNA or block its translation • over 600 miRNAs in humans • regulate at least 1/3 of all protein coding genes in humans • miRNAs are made by RNA polymerase II & are capped and polyadenylated – large and have complex secondary structure that needs to be processed • pre-miRNA molecules are exported out to the cytoplasm Hairpin miRNA 5 3 (a) Primary miRNA transcript MicroRNAs • large pre-miRNA molecule is exported out to the cytoplasm • requires processing • a protein called Dicer cleaves the premiRNA into the mature miRNA • one strand of miRNA associates with proteins RNA-induced silencing complex (RISC) Hairpin Hydrogen bond miRNA Dicer 5 3 (a) Primary miRNA transcript miRNA RISC mRNA degradedTranslation blocked (b) Generation and function of miRNAs MicroRNAs • the RISC base pairs with its complementary mRNA nucleotides – usually in the 3’UTR • if base pairing is extensive – cleavage of mRNA – happens in plant cells • if base pairing is limited – repression of translation – happens in animal cells