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Date Created: 10/15/16
METHODS_DNA CLONING AND ANALYSIS Exon duplication - Different genes can be created by exon duplication via recombination - Homologous noncoding sequences called L1 long interspersed elements lie 5’ and 3’ of the gene and between exons 2 and 3 - Exon duplication by unequal crossing - Homologous recombination between the two parental chromosomes generate one recombinant chromosomes o Gene 1 now has 4 copies of exon instead of three (two copies of exon 3) o Gene 2 is missing 1 exon (exon 3) Gene duplication - Unequal crossing due to repetitive DNA is involved in gene duplication - Unequal crossing over - Each parental chromosome contains one ancestral β-globin gene - Unequal recombination between L1 elements and subsequent independent mutations yield duplicated genes on one chromosome that might encode slightly different proteins Exon shuffling - An evolutionary process for creating new genes form preexisting ones by recombination between introns of two separate genes or by transportation of mobile DNA elements o Mobile DNA elements any DNA sequence that can move to a new position by transportation - Repetitive DNA is involved in “exon shuffling” by enabling gain or loss of exons - A double crossover recombination between two sets of Alu repeats result in an exchange of exons between two genes - This is a higher eukaryote evolution - Recombination between mobile DNA elements (e.g., Alu elements) in introns of two separate genes generated new genes with novel combinations of preexisting exons (e.g., tissue plasminogen activator, the Neu receptor, and epidermal growth factor – all contain an EGF domain) Comparative genomics - The number of protein coding genes in an organism’s genome is not directly related to its biological complexity - The relative proportion of functionally related genes is similar among all eukaryotes - Sequencing of entire genomes from different organisms provides insights into their: o Evolution o Genome organization o Gene function Genomics allows comparative sequence analysis and identification of gene function - Proteins with similar functions often contain similar amino acid sequences encoding functional domains - Because of the degeneracy in the genetic code, related proteins invariably exhibit more sequence similarity than the genes encoding them o For this reason, protein sequences, rather than the corresponding DNA sequences are usually compared - Human gene NF1 o Mutations in NF1 are associated with the inherited disease neurofibromatosis 1 Multiple tumors develop in the peripheral nervous system, causing large protuberances in the skin o NF1 protein similar to Ira o Ira is a GTPase-activating protein (GAP) that activates the GTPase activity of the Ras monomeric G protein that stimulates cell division, turning Ras off. Defective NF1 could cause abnormally high Ras signaling leading to excessive cell division and formation of the tumors o Active Ras promotes cell division o Inactive Ras stops cell division Coding and noncoding human DNA - 98.5% of genomic DNA in higher eukaryotes is “non-coding” - The existence of introns enables the evolution of complex genes (and multidomain proteins) Recombinant DNA Technology - Recombinant DNA Technology is used in DNA cloning - ->DNA Vector + DNA fragment - ->Recombinant DNA - ->Replication of recombinant DNA within host cells - ->Isolation, sequencing and manipulation of purified DNA fragment - Recombinant DNA o Any DNA molecule composed of sequences derived from different sources - Vectors o Used to carry a cDNA or fragment of genomic DNA into a host call for the purpose of gene cloning - Restriction enzymes and DNA ligases facilitate production of recombinant DNA molecules Manipulating DNA – PCR - polymerase chain reaction - In a tube, mix o DNA + 2 oligo primers + 4 dNTPs + DNA polymerase - Then o Denature DNA (95 C) o o Cool to 60 C to allow primers to anneal to DNA o o Raise to 72 C to allow DNA (Taq) polymerase to synthesize DNA (Elongation) o Repeat cycle 40 times Manipulating DNA- The uses of restriction enzymes - Restriction enzymes o Endonucleases produced by bacteria that recognize restriction sites and cleave both DNA strand at restriction sites - DNA fragment cloning/gene subcloning - Southern blotting - Restriction fragment length polymorphism (RFLP) analysis - Gene and fragment mapping - Promoter region analysis - Restriction enzymes make staggered cuts in the two DNA strands at restriction sites, generating fragments that have a single-stranded “tail” at both ends - Restriction enzymes are used extensively because they cut an isolated piece of DNA in the same way each time, reproducibly and predictably Manipulating DNA- Ligation of DNA fragments by DNA ligase - DNA cut by Restriction enzymes can be “ligated” to other DNA sequences by an enzyme called DNA ligase - DNA fragments with sticky or blunt ends can be inserted into vector DNA with the aid of DNA ligase - DNA ligase catalyzes the end to end joining (ligation) of short fragments of DNA - For DNA cloning, purified DNA ligase is used to covalently join the ends of a restriction fragment and vector DNA that have complementary ends. DNA “vectors” allow transfer and cloning of DNA molecules - Plasmids o Circular, double-stranded DNA (dsDNA) molecules that replicate separately from a cell’s chromosomal DNA o Plasmid DNA is duplicated before every division o The extrachromosomal DNA exist in a symbiotic relationship with their host cell - Shuttle vectors: plasmids that can replicate in more than one host organism, e.g. Bacteria & Yeast or Bacteria & Mammalian cells see 5-17. - Properties o Replication origin A marker that permits selection Bacterial Mammalian Yeast Insect o Positive Selection Marker Antibiotic Resistance Auxotrophic complementation o Multiple Cloning sites (MCS) PCR - polymerase chain reaction - can be used for cloning a specific region of DNA - Forensics analysis, rapid screening/cloning of phage/plasmid inserts, cloning specific DNA/RNA sequences, mutation generation, probe generation, sequencing reactions, etc Cloning DNA - Vector and inserts are first cut with the same restriction enzyme - Transformation of competent cells - Positive selection - Each colony is derived from a single cell that took up one plasmid molecule---this is a bacterial clone---the DNA is a DNA clone Manipulating DNA- Transient and stable transfections - Transfection o Introduction of foreign DNA into cells in culture, usually followed by expression of genes in the introduced DNA o Two types Transient Stable - Transient o Vector is NOT integrated into the genome of host cells, and therefore can be lost during cell division - Stable o Vector is STABLY integrated into the genome of host cells, and therefore cannot be lost during cell division Identification of a specific “clone” - Take a membrane -> lift some cells into nitrocellulose filter -> incubate filter in alkaline solution -> hybridize with labeled probe - This technique is based on the ability of nucleic acids to specifically hybridize with one another. Green Fluorescent Protein (GFP) - Proteins fused to Green Fluorescent Protein (GFP) allow their visualization in cells or tissue within animals - ODR10 ORF GFP codes for a fusion protein that can localize to the tip DNA METHODS Getting the lagging strand “up to speed” - The sliding clamp loader and its flexible to proteins allow three polymerases (1 for leading and 2 for lagging strand) to function closely (and coordinately). Microarray - Consists of an organized array of thousands of individual, closely packed, gene-specific sequences attached to the surface of a glass microscope slide - Microarray analysis allows gene expression detection on a whole genome level o Red dye – serum present o Green dye – serum not present o Mix red and green dye o Measure green and red dye fluorescence Red spot – expression of that gene increases in cells after serum addition Green spot – expression of the gene decreases in cells after serum addition - Microarrays data helps identify clusters of co-regulated genes In situ hybridization - RNA in situ hybridization allows detection of gene expression in whole tissues - In situ hybridization can detect activity of specific genes in whole or sectioned embryo Electrophoresis - A technique for separating molecules in a mixture under the influence of an applied electric field. Gel electrophoresis - A tool to separate DNA molecules of different length - Near neutral pH, DNA molecules carry a large negative charge. - In gel electrophoresis, -vely charged DNA moves toward the +ve electrode - The gel restricts the movement of different DNA molecules based on their lengths and charge SDS-polyacrylamide gel electrophoresis - Separation of proteins by SDS-PAGE depends on the size of the protein. Because charge: mass ratio is constant due to binding of SDS to proteins - When a mixture is placed in a gel and electric current is applied, the gel acts as a sieve, allowing smaller species to maneuver more rapidly through its pores than larger species do o Denature proteins with sodium dodecyl sulfate o Place mixture of protein on gel; apply electric field o During electrophoresis, the SDS protein complexes migrate through the polyacrylamide gel o Stain to visualize separated bands Hybridization - Depends on the ability to complementary single-stranded DNA or RNA molecules to associate (hybridize) specifically with each other via base pairing - Hybridization techniques permit detection of specific DNA fragments and mRNAs Southern Blotting - First hybridization technique developed to detect DNA fragments of a specific sequence - Capable of detecting a single specific restriction fragment in the highly complex mixture of fragments produced by cleavage of the entire human genome with a restriction enzyme - Restriction fragments present in the gel are denatured with alkali and transferred onto a nitrocellulose filter or nylon membrane by blotting - The filter is then incubated under hybridization conditions with a specific labeled DNA probe, which is usually generated from a cloned restriction fragment - The DNA restriction fragment that is complementary to the probe hybridizes to it, and its location on the filter can be revealed by autoradiography or by fluorescent imaging Northern Blotting - Similar to southern blotting - An RNA sample is denatured by treatment with an agent such as formaldehyde. The individual RNAs are separated according to size by gel electrophoresis and transferred to a nitrocellulose filter to which the extended denatured RNAs adhere - The filter is then exposed to a labeled DNA probe that is complementary to the gene of interest - Then the labeled filter is subjected to autoradiography Western blotting - For detecting specific proteins - Proteins separated on gel are first transferred to a membrane. - The membrane is exposed to an antibody specific to a protein of interest. - Antibody-antigen complexes are then detected by secondary antibody and fluorescence or colorimetric assays 2-Dimensional gel electrophoresis - Proteins are separated first by their charges and then by their masses Centrifugation - Can separate particle and molecules that differ in mass and density - There are two major types of centrifugation: - Differential centrifugation o Molecules or organelles sediment (travel) through a solution at a certain rate, eventually reaching the bottom. - Density gradient (rate-zonal) centrifugation o Molecules travel in a gradient (of sucrose) and find their own isopycnic (=equilibrium) density and remain there. Protein purification by chromatography - Gel filtration o separates on basis of size and shape - Ion – exchange o separates on the basis of net charge - Affinity chromatography o depends on the unique ability of a ligand attached to the column matrix to bind to a protein DNA REPLICATION Three Models of DNA Replication - Dispersive o New double helix: A composite of Parent and Daughter regions - Conservative o The two daughter strands would form a new double-stranded DNA molecule and the parent duplex would remain intact o Would never generate H-L DNA (Heavy-light) (hybrid DNA) - Semi conservative o The parent strands would be permanently separated and each would form a duplex molecule with the newly synthesized daughter strand base-paired to it o DNA replication is semi conservative Parent strand –> heavy Generation 1 –> 2 Heavy – Light Generation 2 –> 2 Heavy – Light and 2 Light DNA is synthesis occurs only in the 5’ to 3’ direction - Directionality of DNA (antiparallel strands) has consequences for DNA synthesis, because DNA polymerase can synthesize DNA in only one direction by adding nucleotides to the 3' end (i.e. extending the 3’ end) DNA polymerase cannot initiate chain synthesis de novo - They require a short, preexisting RNA or DNA strand, called a primer, to begin growth - With a primer base-paired to the template strand, a DNA polymerase adds deoxy ribonucleotides to the free hydroxyl group at the 3’ end of the primer as directed by the sequence of the template strand DNA Replication - In order for duplex DNA to function as a template during replication, the two intertwined strands must be unwound or melted to make their bases available for pairing with the bases of the dNTPs that are polymerized into the newly synthesized daughter strands - The unwinding of the parent DNA strands is performed by enzymes called helicases - Unwinding begins at segments in a DNA molecule called replication origins or simply origins - Once helicases have unwound the parent DNA at an origin, a specialized RNNA polymerase called primase forms a short RNA primer complementary to the unwound template strands - The primer is elongated by DNA polymerase α for another 25 nucleotides or so, forming a primer made of RNA at the 5’ end and DNA at the 3’ end Replication Fork - The DNA region at which all proteins come to together to carry out the synthesis of daughter strands - As replication proceeds, the replication fork and the associated proteins move away from the origin - Local unwinding of duplex DNA produces torsional stress, which is relieved by topoisomerase I o In order for DNA polymerase to move along and copy a duplex DNA, helicase must sequentially unwind the duplex and the topoisomerase must remove the supercoils that form - Synthesis of one daughter strand, called the leading strand, can proceed continuously from a single RNA primer in the 5’ to 3’ direction, the same direction as movement of the replication fork - Problem in synthesis of the other daughter strand, lagging strand o Growth of the lagging strand must occur in the 5’ -> 3’ direction, copying of its template strand must somehow proceed in the opposite direction from the movement of the replication fork o A cell accomplished this feat by synthesizing a new primer every 100 to 200 nucleotides on that template strand as more of the strand is exposed by unwinding o Each of these primers, base-paired to the template strand, is elongated in the 5’ -> 3’ direction, forming discontinuous segments named Okazaki fragments o The RNA primer of each Okazaki fragment is removed and replaced by DNA chain growth from the neighboring Okazaki fragment o Finally, an enzyme called DNA ligase joins the adjacent fragments The challenges of replicating DNA 1. DNA needs to be unwound, i.e. the two strands need to be separated. Answer: Initiator, Helicase proteins 2. The separated DNA strands need to be kept separated for some time. Answer: SSB Proteins (Single-Strand Binding Proteins) 3. Supercoils induced in upstream DNA need to be removed. Answer: Topoisomerase, gyrase proteins 4. DNA cannot be synthesized without a “primer” sequence. Answer: Primase protein 5. DNA needs to be synthesized by adding nucleotides… Answer: DNA Polymerase protein complex Bidirectional DNA replication mechanism - In theory, DNA replication from a single origin could involve one replication fork that moves in one direction - Alternatively, two replication forks might assemble at a single origin and then move in opposite directions, leading to directional growth of both daughter strand - Eukaryotic DNA contains multiple replication origins separated by tens to hundreds of kilobases. - A six-subunit protein origin recognition complex (ORC) binds to each replication origin and associates with other proteins required to load two hexameric helicases, composed of six homologous MCM proteins, oriented in opposite directions. RPA proteins bind to separated parent strands at the origin. - Replication at the origins is coordinated by DDK (kinase) activation of MCM helicase. DDK is regulated by S-phase cyclin-dependent kinases. - Two large T-antigen hexameric helicases bind at the replication origin in opposite orientations. • Step 1: Helicases use ATP hydrolysis energy to move in opposite directions, unwinding the parent DNA and generating single-stranded templates, which are bound by RPA proteins. • Step 2: Primase–Pol α complexes synthesize short primers (red) base-paired to each of the separated parent strands. • Step 3: PCNA-Rfc–Pol ε complexes replace the primase–Pol α complexes and extend the short primers, generating the leading strands (dark green) at each replication fork. • Step 4: The helicases continue to unwind the parent strands, and RPA proteins bind to the newly exposed single-stranded regions. • Step 5: PCNA-Rfc–Pol ε complexes extend the leading strands. • Step 6: Concurrently, primase–Pol α complexes synthesize primers for lagging- strand synthesis at each replication fork. • Step 7: PCNA-Rfc–Pol δ complexes displace the primase–Pol α complexes and extend the lagging-strand Okazaki fragments (light green), which are ligated to the 5′ ends of the leading strands. - Unwinding and synthesis of leading and lagging strands occur concurrently SV40 DNA - Circular genome of a virus that infects monkeys - The molecular machine that replicates SV40 DNA contains only one Viral protein; all other proteins involved in SV40 DNA replication are provided by the host cell - The viral protein is large T antigen - Forms a hexameric replicative helicase - A protein that uses energy from ATP hydrolysis to unwind the parent strands at a replication fork - Primers for the leading and lagging daughter strands are synthesized by a complex of primase, which synthesizes a short RNA primer and DNA polymerase α (Pol α) o (Pol α) extends the RNA primer with deoxy ribonucleotides for another 25 nucleotides or so, forming a mixed RNA and DNA primer - The primer is extended into daughter-strand DNA by DNA polymerase δ (Pol δ) o (Pol δ) is less likely to make errors during copying of the template strand than is Pol α because of its proofread mechanism - During the replication of cellular DNA Pol δ synthesis lagging strand DNA, while DNA polymerase £ (Pol £) synthesis most of the length of the leading strand - Pol δ and Pol £ each form a complex with PCNA (proliferating cell nuclear antigen) following primer synthesis - PCNA o A homo-trimeric protein that has a central hole through which the daughter duplex DNA passes, thereby preventing the PCNA-Pol δ and PCNA-Pol £ completes from dissociating from the template - RFC o Replication factor C o A pentameric protein o Opens the PCNA ring so that it can encircle the short region of double-stranded DNA synthesized by Pol α - Leading strand is extended by Pol £ which can extend the growing strand up to the replication fork - Lagging strand is bound by multiple copies of RPA (replication protein A) o A heterotrimeric protein o Maintains the template in a uniform conformation that is optimal for copying by Pol δ - Bound RPA proteins are dislodged from the protein strand by Pol δ as it synthesizes the complementary strand base paired with the parent strand DNA REPAIR AND TRANSCRIPTION Maintenance of histone H3 lysine 9 methylation during chromosome replication - Heterochromatin epigenetic code memory: • When DNA in heterochromatin is replicated, histone octamers di- or trimethylated at H3 lysine 9 are distributed to both daughter chromosomes along with an equal number of newly assembled histone octamers. • The H3K9 HMT associated with the H3K9 di- and trimethylated nucleosomes methylates lysine 9 of the newly assembled nucleosomes, regenerating the heterochromatin in both daughter chromosomes. - Mechanism o Parent (old) histones randomly associate with the two daughter chromosomes. o Unmethylated (new) histones synthesized during S phase also assemble in both daughter chromosomes. o Histone H3 lysine 9 methyl transferases (H3K9 HMT) associate with parent nucleosomes bearing histone 3 lysine 9 di- or trimethylation marks methylate the newly added unmodified nucleosomes o Maintains histone H3 lysine 9 methylation marks during repeated cell divisions, unless they are specifically removed by a histone demethylase The End-replication problem - As the replication fork approaches the end of a linear chromosome, synthesis of the leading strand continues to the end of the DNA template strand, completing one daughter strand - However, because the lagging strand template is copied in a discontinuous fashion, it cannot be replicated in its entirety - When the final RNA primer is removed, there is no upstream strand onto which RNA polymerase can build to fill the resulting gap - Without some special mechanism, the daughter DNA strand resulting from lagging strand synthesis would be shortened at each cell division - This problem is solved by an enzyme that adds telomeric repeat Telomerase - Addition of telomeric sequences by Telomerase prevents shortening of chromosomes - Telomerase function in 3’ end extension and completion of lagging strand synthesis by DNA Pol - 1. The telomerase contains an RNA template that base pairs to the 3’end of the lagging strand template o The telomerase catalytic site then adds deoxyribonucleotides TTG, using the RNA as a template - 2. The strands of the resulting RNA-DNA duplex are then thought to slip (translocate) relative to each other so that the TTG sequence at the 3’end of the replicating DNA base pairs to the complementary RNA sequence in the telomerase RNA - 3. The 3’ end of the replication DNA is then again extended by telomerase - DNA polymerase α-primase can prime synthesis of new Okazaki fragments on this extended template strand - The net result prevents shortening of the lagging strand at each cycle of DNA replication DNA Repair and Recombination - Damage to DNA is unavoidable - Damage can be cause by o Spontaneous cleavage of chemical bonds in DNA o Environmental agents, such as ionizing radiation o Reaction with genotoxic products of normal cellular metabolism or occur in the environment - DNA damage can cause DNA mutation, a change in the normal DNA sequence - Mutation o A change in the normal DNA sequence o Can occur during replication when a DNA polymerase inserts the wwrong nucleotide a it reads a damaged template o Occur at a low frequency as the result of copying errors introduced by DNA polymerases when they replicate an undamaged template DNA Polymerase has proofreading activity - Proofreading depends on the 3’ -> 5’ exonuclease activity of some DNA polymerases - Proofreading by DNA polymerase repairs damage/copying errors. - DNA polymerases have a 3D structure resembling a half-opened right hand. The “fingers” bind the single-stranded segment of the template strand, and the polymerase catalytic activity (Pol) lies in the junction between the fingers and palm. - Incorrect base addition at the 3′ end causes melting of the newly formed end of the duplex and polymerase pausing. - The growing strand 3′ end is transferred to the 3′ → 5′ exonuclease site (Exo) about 3 nm away, where the mispaired base is removed. - The 3′ end flips back into the polymerase site and elongation resumes Spontaneous deamination results in point mutation - A protein coding region point mutation can introduce a stop codon (nonsense) or a change in protein amino acid sequence (missense), or may not change the amino acid sequence (silent, usually in the third codon position, e.g., GAG to GAA; both encode glutamine). - A gene regulatory region point mutation can change gene expression. - 5-methylcytosine (C) deamination to thymine (T) is a frequent cause of point mutations; if C is not restored, the normal C∙G base pair will become a T-A mutation during replication of one of the DNA strands Repair of TG mismatches by Base Excision Repair - Occurs before DNA replication - The GT mismatch is recognized by a DNA glycosylase that flips the thymine base out of the helix and then hydrolyzes the bond that connects it to the sugar-phosphate DNA backbone - An endonuclease specific for the resultant abasic site (apurinic endonuclease 1, APE1) then cuts the DNA backbone - The deoxyribose phosphate is removed by an endonuclease, apurinic lyase (AP lyase) associated with DNA polymerase β, a specialized DNA polymerase used in repairs - The gap is then filled in by DNA Pol β and sealed by DNA ligase, restoring the original GC base pair Mismatch Excision Repair - Repairs other mismatches and small insertions and deletions - Occurs after DNA replication - A complex of MSH2 and MSH6 covers the mismatch site - This binding triggers binding of MLH1 and PMS2. - The activity of these proteins (endonuclease cuts the daughter strand, helicase unwinds the DNA, exonuclease nucleotides) together results in the removal of several nucleotides on both sides of the mismatch specifically only on the newly made daughter strand - The gap is then repaired by DNA Pol δ and DNA ligase Nucleotide Excision Repair - Mutations in several XP genes such as XP-G genes inactivate this repair system, causing xeroderma pigmentosum, a hereditary predisposition to UV-induced melanoma and squamous cell carcinoma skin cancers - UV irradiation causes formation of carbon-carbon bonds between adjacent thymines in DNA (thymine dimers), which interfere with replication and transcription - Excision-repair mechanism recognizes the T-T dimer-caused distortion in the DNA - XP-C (xeroderma pigmentosum C protein)-23B protein complex recognizes double helix distortion - TFII H then binds, followed by RPA and XP-G. Together, they melt ~25bp of DNA - XP-G and another endonuclease (XP-F) cut the damaged strand about 24-32 apart on each side of the lesion - DNA polymerase and ligase close the gap Two systems Use Recombination to Repair Double Strand Breaks in DNA - Ionizing radiation and some anticancer drugs cause double-strand breaks in DNA - These lesions are particularly severe because incorrect rejoining of double strands of DNA can lead to gross chromosomal rearrangement that can affect the functioning of genes - Two types o Homologous recombination o Non-homologous recombination - Non-Homologous End Joining” repairs double stranded (DS) breaks o DS breaks (which can be caused by ionizing radiation or anticancer drugs) can be repaired by homologous recombination. o When a sister chromatid is not available, DS breaks are repaired by this pathway. The broken ends are joined together but there is a loss of several nucleotides of DNA o A complex of KU80, KU70 and DNA PK (protein kinase) binds to each broken end o Other repair proteins such as nucleases surgically remove DNA near the break o This repair mechanism is error prone: base loss causes gene mutation, and incorrect rejoining can cause gross chromosomal rearrangement mutations that affect the expression of genes or create a “hybrid” gene that encodes the N- terminal portion of one amino acid sequence fused to the C-terminal portion of a completely different protein Nobel Prize in Chemistry 2015 - Tomas Lindahl o base excision repair - Aziz Sancar o nucleotide excision repair - Paul Modrich o mismatch repair TRANSCRIPTION Transcriptional control of gene expression is critical for proper functioning of cells - Controlling transcription allows the cell to achieve the following: o Express select genes in all the cells most of the time – termed constitutive expression (e.g. rRNA, actin, genes involved in cellular metabolism) o Express select genes in specific cells – termed tissue/cell specific expression (e.g. Pax6 is expressed in cells of the eye, brain, pancreas) o Express select genes in many cells at a particular time in development (e.g. Shh gene is expressed in cells of the notochord early in development) o Express select genes based on its environment (e.g. Genes encoding heat shock proteins are expressed (upregulated) in conditions of stress) Simple and complex transcription units - Gene o The entire DNA sequence required for the synthesis of a functional protein or RNA molecule. - A gene includes: o The coding regions o The non-coding introns o The non-coding control regions (e.g. promoters, enhancers) - Cap site - Splice site - Poly (A) site - Promoter site Overview of eukaryotic transcription control - One of the DNA strands in a gene is the template for transcription of an RNA by complementary base pairing o RNA polymerase catalyzes phosphodiester bond formation between the 3′ oxygen of the growing strand and the α phosphate of a correctly base-paired rNTP (nucleophilic attack by the 3′ oxygen in the growing RNA chain on the α phosphate of the next nucleotide precursor to be added) – always in the 5′→3′ direction. - Inactive genes are assembled into regions of condensed chromatin that inhibit RNA polymerases and their associated general transcription factors from interacting with promoters - Many activators of transcription and chromatin remodelers are needed at several places to turn a gene “on” by changing its state from a condensed, inactive hetero-chromatic state into “loosened” euchromatin - hetero-chromatic o closed - euchromatin o open - During transcriptional activation, Poll II nucleotides due to the action of the elongation inhibitor NELF associated with DSIF Co-activators - chromatin/histone modifying proteins - Activator-directed histone acetylation - Facilitate euchromatin formation (open, de-condensed) - Facilitates access of general TFs for transcription initiation - Hyper-acetylation of histones N-terminal tails - Histone Acetylase Complex catalytic subunit o Causes acetylation of histones o SAGA complex - Co-activators recruited by activation domain (AD) of Gcn4 - The DNA-binding domain of the activator Gcn4 interacts with specific upstream activating sequences (UAS) of the genes it regulates. The Gcn4 activation domain (AD) then interacts with a multiprotein histone acetylase complex that includes the Gcn5 catalytic subunit - Co-activators can be histone modifying enzymes like Gcn5, or can be chromatin remodeling complexes, altering the spacing of nucleosomes on DNA or changing the structure of chromatin in other ways. Repressors - Also modify histone proteins - Facilitates heterochromatin formation (closed, condensed) - Inhibits access of general TFs for transcription initiation - Repressor-directed histone deacetylation - Repressor domains on repressor proteins can recruit silencing complexes (function opposite to that of mediators) or co-repressors (function opposite to that of co- activators) - Deacetylation of histones N-terminal tails - Co-repressor is recruited by the repressor domain (RD) of Ume6 - Histone deacetylase Complex o Rpd3L Methylation of histones - Can also interfere with transcription by changing the structure of chromatin - Methylation of H3 Lys 9 can compact chromatin and silence it - HP1 (heterochromatin protein 1) contributes to heterochromatin condensation: o binds to histone H3 N-terminal tails trimethylated at lysine 9 o associates with other histone-bound HP1 molecules - Heterochromatin condensation can spread along a chromosome: o HP1 chromoshadow domain binds a histone methyltransferase (H3K9 HMT) that methylates lysine 9 of a histone H3 in an adjacent nucleosome, which creates a binding site for another HP1 on the neighboring nucleosome. o The spreading process continues until it encounters a “boundary element” where several nonhistone proteins are bound to the DNA Basic features of eukaryotic transcription - The nucleotide position where RNA polymerase begins transcription of a gene is designated +1. o The polymerase travels “downstream” toward the 3’ end on the DNA, and downstream bases are designated with positive numbers. o “Upstream” bases are designated with negative numbers. o Important gene features lie upstream of the transcription start site, including promoter sequences recognized by transcription factors that recruit RNA polymerase to the gene. - The DNA strand being transcribed is the template strand; its complement is the nontemplate strand. - The RNA synthesized is complementary to the template strand and is therefore identical with the nontemplate strand sequence, except with uracil in place of thymine. Structure of a mammalian gene - Three types of promoters - TATA boxes o ≈-31 to -26 o First to be sequenced and studied through in vitro transcription system - Initiator Sequences o -2 to +4 o Most naturally occurring intiator elelments have a cytosine (C) at the -1 position and an adenine (A) residue at the transcription that site (+1) - CpG Islands o +28 to +32 o P represents phosphate between the C and G nucleotides o CpG promoters are Present in 60-70% of protein coding genes. Generally, encodes “Housekeeping Genes” that are not transcribed at high levels Enhancers - A regulatory sequence in eukaryotic DNA that may be located at a great distance from the gene it controls or even within the coding sequence - Can work at a distance because DNA is flexible Composition, similarities and differences among RNA polymerases - Bacteria (1 RNA polymerase): o w factor + core enzyme - Eukaryotic o Three - RNA Pol I o Examples rRNAs 28S, 5.8S 18S o β and β’ present o αI and αII present (same as RNA Pol III) o ω-like subunits present o 4 common subunits o Addition enzyme specific subunits +5 - RNA Pol II o Examples Pre-mRNA -> mRNA mRNA snRNA siRNA miRNA o β and β’ present o CTD attached to β’1 o αI and αII present (not the same shape as RNA Pol I and RNA Pol III) o ω-like subunits present o 4 common subunits o Addition enzyme specific subunits +3 - RNA Pol III o Examples tRNAs 5S rRNA snRNA U6 7S RNA o β and β’ present o αI and αII present (same as RNA Pol I) o ω-like subunits present o 4 common subunits o Addition enzyme specific subunits +7 RNA Pol II Beta1 subunit has an important C terminal domain (CTD) - The Carboxyl end of the RNA Pol II B1 subunit has a sequence of seven amino acids that is repeated multiple times. - Heptapeptide repeat consensus - o Tyr-Ser-Pro-Thr-Ser-Pro-Ser - CTD = C terminal domain - Red – Ab against phosphorylated CTD (active transcription) - Green – Ab against non-phosphorylated CTD (not active transcription) Transcription - Initiation o RNA polymerase, with the help of initiation factors recognize and binds to a specific sequences of double-stranded DNA called a promoter o After binding, RNA polymerase and the initiation factors separate the DNA strands o ≈12 – 14 base pairs of DNA around the transcription start site on the template strand are separated, which allows the template strand to enter the active site of the enzymes o The 12-14 base pair region of melted DNA in the polymerase is known as the transcription bubble o Transcription initiation is considered complete when the first two ribonucleotides of an RNA chain are linked by a phosphodiester bond o After several ribonucleotides have been polymerized, RNA polymerase dissociates from the promoter RNA and initiation factors - Elongation o RNA polymerase moves along the template DNA, opening the double-stranded DNA o One ribonucleotide at a time is added by the polymerase to the 3’ end of the growing RNA chain o the enzyme maintains a melted region of ≈14 – 20 base pairs in the transcription bubble o ≈8 nucleotides at the 3’ end of the growing RNA strand in the transcription bubble - Termination o The completed RNA molecule is released from the RNA polymerase and the polymerase dissociates from the template RNA o Once it is realized, an RNA polymerase is free to transcribe the same gene or another gene Assembly of general transcription machinery over core promoter - TBP (TATA binding protein) subunit of TFIID is the first protein to bind to a TATA box promoter - This domain of TBP folds into a saddle-shaped structure - TBP interacts with the minor groove in DNA, bending the helix considerable - Once TFIID has bound to the TATA box, TFIIA and TFIIB can bind - TFIIA associates with TBP and DNA on the upstream side of the TBP-TATA box complex - The C-terminal domain of TFIIB makes contact with both TBP and DNA on either side of the TATA box - TFIIB assists Pol II in melting DNA - Following TFIIB binding, a performed complex of TFIIF and Pol II binds, positioning the polymerase over the start site - Then TFIIE binds o Creates a docking site for TFIIH - TFIIH has helicase activity - TFIIH has kinase activity – phosphorylates CTD Ser 5 - Binding of TFIIH completes assembly of the transcription preinitiation complex TRANSCRIPTION – POST TRANSITIONAL CONTROL DNA binding domains can recognize specific DNA sequences - Different nucleotide base pairs can be recognized (by a DNA binding protein) from their edges without the need to open the DNA double helix. - Major groove offers significantly more complex pattern for bonding than minor groove - Surface of DNA binding protein has a complementary fit with the specific surface features of the double helix Transcription activators have various specialized domains - DNA binding domains usually contain one or several DNA binding motifs and a dimerization motif. - Activation domains are protein-protein interacting domains. To induce transcription, they usually bind to proteins called mediators or to co-activators. - Repressors of transcription have a DNA binding domain and a repressor domain that recruits silencing complex or a co-repressor Examples of DNA binding motifs - Helix turn helix o 2 a-helices connected by short extended chain of amino acid - Zn finger o C H2Zinc finger Most common DNA binding motif encoded in the human genome and the genomes of other mammals Cysteine and histidine 3 or more repeating finger units o C Z4nc finger Only two finger units - Leucine zipper o DNA-binding domain of the yeast Gcn4 transcription o Hydrophobic interactions forming a coiled coil structure - p53 o Neither zinc, nor a-helices or b-sheets; Peptide loops bind both major or minor grooves o R248 is frequently found to be mutated in human cancer cells Activators often work in pairs - NFAT and AP1 lower binding affinity to DNA on their own but together, bind to DNA with higher affinity - NFAT and AP1 are structurally unrelated proteins but are neighboring sites - Both bind to IL-2 promoter o Important for precisely controlled immune response (IL = Interleukin) Combinatorial binding establishes a precise level of activation or repression - Transcription factor monomers A, B and C can all interact with one another, creating six different alternative combinations of activation domains that can bind at the same site - An inhibitory factor that interacts only with the dimerization domain of “A” o transcription activated – sites 2, 3, and 6 o transcriptional activation inhibited – sites 1, 4, and 5 - Levels of activators and repressors vary in different tissues Multiprotein complexes assemble on enhancers - Enhancers o 50-200 bp in length o Binding sites for multiple transcription factors - Enhanceosome o Large DNA-protein complexes on an enhancer sequence - Multiple binding sites (for DNA binding proteins) in the b-interferon enhancer generates “combinatorial complexity” in transcription control Mediator complex - Is recruited when it binds to many activators at the same time - A “mediator” is a multiprotein nucleoprotein complex of 100+ polypeptides that forms a molecular bridge between activation domains in TF proteins and Pol II. - Stimulates assembly of pre-initiation complex on promoter - Each protein subunit of a mediator can bind to different types of activation domain on activators. - The function of the mediator is to facilitate the binding of Pol II-TFIIF to TFIID/TFIIB DNA complex bound to the core promoter - The mediator enables integration of signals from several activators at a single promoter RNA Pol II CTD controls transcription and RNA processing - RNA Pol II functions to o Transcribe DNA into RNA o Facilitate pre-mRNA processing to make mRNA - Specific RNA modifying proteins are recruited to phosphorylated RNA Pol II CTD at specific stages of transcription o Capping o Elongation o Splicing o mRNA cleavage and polyadenylation o Termination - Exonuclease (functions to degrade this part of the transcript that is downstream of cleaved mRNA) - De-phosphorylation at Tyr 1 of CTD is necessary for recruitment of termination factors and exonuclease - After transcript cleavage by exonuclease, RNA Pol II dissociates from DNA Addition of a 5’- Cap to eukaryotic mRNAs - A protective cap composed of 7-methylguanosine is added to the 5’ end of ~25 nucleotide long RNA transcript - 5’ cap marks RNA as mRNA precursors and protects them from RNA-digesting enzymes - A multi-subunit Capping enzyme associates with phosphorylated CTD of RNA Pol II, then gets activated - Separate enzymes transfer methyl groups from S-adenosylmethionine to the N7 position of guanine and 2’ position of ribose - The 5’ end of a nascent RNA contains a 5’ triphosphate from the initiating rNTP. The ƴ phosphate is removed in the first step of capping, while the remaining α and β phosphates remain associated with the cap - The third phosphate of the 5’, 5’ triphosphate bond is derived from the α phosphate of the GTP that donates the guanine - The methyl donor for methylation of the cap guanine and the first one or two ribose of the mRNA is S-adenosylmethionine Regulation of transcription elongation - The TAR element in the HIV transcript contains sequences recognized by Tat and the cellular protein cyclin T - Cyclin T activates and helps position the protein kinase CDK9 near its substrates, the CTD of RNA polymerase II, NELF, and DSIF, CTD phosphorylation at serine 2 if the Pol II CTD heptad repeat is required for transcription elongation - The CTD of RNA pol II must remain phosphorylated or transcription will stop - Negative elongation factor, binds to Pol II, leads it to pause transcription after 20-50 nucleotides - Inhibition is relieved when NELF, DSIF and Pol II CTD (serine 2) are phosphorylated by protein kinase Cdk9 - HIV Tat (RNA binding protein) binds to TAR (RNA) which allows Cyclin T/Cdk9 to be recruited, which will phosphorylate the CTD of pol II. This is the mechanism by which the virus ensures RNA Pol II transcription in infected cells. - In normal cells, Cyclin T/Cdk9 is recruited by DNA binding proteins RNA Pol II CTD is the site of assembly for various RNA processing factors e.g. cleavage factors - RNA Pol II CTD when phosphorylated, acts as a site for CPSF and CStF binding. - This is a convenient location for these proteins to function in cleavage and poly- adenylation. Cleavage and polyadenylation of pre mRNA - CPSF binds to the upstream AAUAAA polyadenylation signal - 3 more protein binds to the CPSR-RNA complex - CStF, CFI and CFII - CStF o Interacts with a downstream GU- or U-rich sequence and with bound CPSF, forming a loop in the RNA - CFI, CFII o Helps stabilize the complex - Binding of poly A polymerase (PAP) stimulates cleavage at a poly (A) cleavage site, which is usually 15-20 nucleotides 3’ of the upstream polyadenylation signal - The cleavage factors are released, as is the downstream RNA cleavage product, which is rapidly degraded - Bound PAP then adds about 12A residues at a slow rate to the 3’ -hydroxyl group generated by the cleavage reaction - Binding of nuclear poly A – binding protein (PABRN1) to the initial short poly A tail accelerates the rate of addition by PAP - After 200-250 A residues have been added, PABPN1 signals PAP to stop polymerization
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