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BIOLOGY 2C03 (138)
Joe Kim (16)

Module 7(1-8) - Feb 26-March14 - BIO 2C03

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Joe Kim

BIO 2C03 2013 Module 7 Lecture 1 – Structure of the Genome and Chemical Nature of DNA (February 26) Chapter 10: Chemical Nature of DNA – Structure of the Genome  The genome before 2005 o Most of the genome is non-coding o Most of the genome consists of “junk DNA:  The genome after 2005 o Most of the genome is transcribed o Most of the genome appears to be transcribed into non-coding RNAs (ncRNAs) Gene Expression  The Enhancer – combination of distal regulatory elements with specific properties o Works at great distances (thousands of kb away) o Works in a “position-independent manner” i.e. upstream, downstream, within gene (intron) o Works in an “orientation-independent manner” o Can bend the DNA (through the action of “architectural factors”) o Generally recruit the General Transcription Machinery through Co- Activators including the Mediator  Basic processes of eukaryotic transcription: Formation of the Preinitiation Complex of PIC o Binding of activators promotes the recruitment of Co-Activators and Nucleosome Remodeling Complexes o Chromatin is decondensed as a result of histone modifications and nucleosome “eviction” o GTPs and RNAPII are the recruited to form the PIC or Preinitiation Complex The Dark Genome - ncRNAs  Complexity of transcription around a hypothetical gene 1 BIO 2C03 2013 MicroRNAs and the Control of Translation – RNA Interference  MicroRNAs are non-coding regulatory RNAs  Control of gene expression vs. degradation by RNA interference  Figure: regulation by microRNA (control of gene expression) left side; regulation by dsRNA (RNA Interference) right side Cancer Genetics – Personalized Medicine  Towards personalized medicine  All patient with same diagnosis  genetic chip  1) non responders and toxic responders; treat with alternative drug or dose 2) Responders and patients not predisposed to toxicity; treat with conventional drug or dose Introduction to Biological Macromolecules – DNA and RNA 2 BIO 2C03 2013  DNA is highly structured  RNA is highly structures  Chromatin 3 BIO 2C03 2013 Encoding Genetic Information  The genetic code: o Requires sufficient complexity to determine all traits of the organism o Must be copied accurately and transmitted faithfully o Must be “interpretable” or “translatable” to provide all traits Biological Macromolecule Carrying Genetic Code  Proteins with their 20 types of amino acids could generate a greater variety of molecular species and appeared more complex  Nucleic Acids (DNA, RNA) were made of only four repeated units (nucleotides)  Different organisms have DNA with different base composition  Chargaff’s Rule: The amount of adenine (A) is always equal to the amount of thymine (T) and the amount of guanine (G) is equal to the amount of cytosine (C); (Erwin Chargaff, 1948); A=T, G=C DNA as the “Transforming Principle”  The discover of the “Transforming principle” by Fred Griffith (circa 1928) would eventually lead to the idea that DNA is the source of genetic information  DNase but not RNase or Proteases destroys the Transforming Principle (Avery, Macleod, McCarty) 4 BIO 2C03 2013  The use of bacteriophage as model systems – Hershey-Chase experiment #1  Discovery of the Structure of DNA (Crick, Watson, Ashbury, Franklin, Wilkins, 1947-1953) o Ashbury and then Franklin-Wilkins generate the fist diffraction pattern of DNA 5 BIO 2C03 2013 o Using “molecular modeling”; Watson-Crick determined that:  Adenine can bind with Thymine and Guanine with Cytosine when in a double helix, the sugar phosphate backbone on the outside and the bases on the inside of the helix o The double helix model of Watson-Crick  Consistent with the diffraction patterns obtained by Franklin-Wilkins  Explains Chargaff’s rule  Suggests a mechanism for DNA replication  Nobel Prize in Sweden, 1962 RNA as Genetic Material: Beyond the Central Dogma  Heintz Fraenkel-Contrat demonstrated in 1957 that the Tobacco Mosaic Virus (TMV) contains RNA and proteins but no DNA  Showed that RNA is the genetic material in TMV  Retroviruses also carry an RNA genome  A Rous sarcoma virus (named after Peyton Rous) 6 BIO 2C03 2013 DNA Chemistry 101  Four nitrogenous bases form the nucleotides in DNA and RNA  Deoxyribonucelotides Key Features of the DNA double helix  The two polynucleotide strands are antiparallel  The sugar-phosphate backbone is on the outside and is formed through covalent phosphodiester bonds  The two DNA strands are held together through hydrogen bonding of bases located in the inside  Bonding can occur between A and T, G and C  The two strands are not identical but are complementary Lecture 2 – Structure of the Genome – Genome 101 (February 28) Chapter 11 DNA Exists in a Highly Compacted Form  The degree of folding (condensation, packaging) of DNA variesdepending on the region of the genome and phase of the cell cycle  For instance, highly transcribed genes are found in a relatively “relaxed” state during interphase while silent regions are highly condensed  DNA is highly condensed in chromosomes during mitosis  Euchromatin undergoes condensation and decondensation during the cell cycle (eukaryotes)  Heterochromatin contains DNA that is always condensed (eg/ the Barr body, centromeres, telomerase) (eukaryotes)  In placental mammals – random X chromosome inactivation generates a highly condensed structure in interphase known as the Barr body of female cells The Bacterial Chromosome  Bacterial chromosome o Singular DNA molecule o Found in a highly organized structure localized in the nucleoid (a region in the cytoplasm) 7 BIO 2C03 2013  Condensation will depend on the degree of “supercoiling” of the DNA and the interaction with specialized proteins  Topoisomerases – induce or relieve supercoiling by breaking and rotating the DNA strands before rejoining the broken ends  Supercoiling is important for condensation of the DNA  Negative supercoiling promotes strand separation, facilitating DNA replication and transcription Eukaryotic Chromosome  Chromatin Structure 1. Histones – small proteins; 12-35kDa  Rich in arginine’s and lysine’s (positively charged)  Make contact with the phosphates of DNA (negatively charged)  DNA wraps around a core of 8 histones forming the Nucleosome  Some are highly modified at the N-terminus by acetylation, methylation, phosphorylation, ubiquitinylation (in gene regulation) 2. Non-Histone Chromosomal Proteins (“Architectural Factors)  About 50% of the protein mass of chromosomes  Eg/ HMG (High Mobility Group) Proteins  Mall  Bind DNA often in A-T rich regions, inducing DNA “bending” and DNA looping  Mutations in HMG protein genes have been found in some benign tumors (eg/ lipomas) 8 BIO 2C03 2013  Important for transcription, the function of “Enhancers”, DNA replication, repair and recombination 3. Chromosomal Scaffold Proteins – structural components of chromosomes  Contribute to chromatin folding and chromosome packing 4. Structural components of the kinetochore – involved in chromosome attachment on the spindle and chromosome movement during mitosis 5. The Shelterin Complex – Caps and protects the telomeres 6. Proteins and enzymes or the DNA replication machinery  DNA Polymerases  DNA helicases  RNA primases etc 7. Proteins involved in gene expression  Transcription factors  Chromatin modifying factors (eg/ histone acetyl transferases)  Chromatin remodeling complexes  RNA polymerases and “basic transcription machinery”  Nucleosome – basic unit in chromatin formation; core particle o Consists of 145-147 bp of DNA wrapped ~2x around an octamer of 8 histones including 2 copies each of H2A, H2B, H3 and H4 9 BIO 2C03 2013 o The fifth histone (H1) binds to about 20-22 bp of DNA where the DNA enters and leaves the core histones, acting as a clamp to stabilize the core particle o The core particle and associated histone H1 contains about 167 bp of DNA  forms the Chromatosome o Chromatosomes are located at regular intervals and are separated by about 30-40 bp of DNA referred to as the “linker DNA” – provides a “beads-on-a-string” conformation of chromatin o The linker DNA is relatively less protected and mis more susceptible to the action of nucleases than DNA associated with histones o Limited DNase digestions of chromatin generates a typical ladder pattern when analyzed on agarose gel  Higher-Order Chromatin o Chromatin can be further condensed to form helical fibers of about 30 nm in diameter o The 30-nm fibers can form loops of DNA that can be further condensed to eventually form a chromatid of circa 700 nm in width o Figure – Right; EM view of 30-nm fiber; Left; Schematic representation o Chromosomal puffs are region of “relaxed” chromatin and gene expression o Figure – giant polytene chromosomes in salivary glands of Drosophila larvae  Centromere o The centromere is recognized by the kinetochore o Kinetochore – protein complex responsible for the attachment of chromosomes to the spindle and the movement of chromosomes at mitosis and meiosis  Protein of the kinetochore are responsible for the control of the “Spindle Checkpoint”  Spindle Checkpoint – inhibits chromosome segregation until all chromosomes are properly attached on the spindle  Proteins of the kinetochore provide the signal to block chromosome segregation during the Spindle Checkpoint o A broken chromosome is not segregated properly if it does not include a centromere o Sequences of the centromere vary considerably in different organisms o In yeast – the centromere contains a sequence repeated multiple times; centromere functions autonomously and is sufficient to promote segregation of a unrelated DNA molecule (like a plasmid) o In higher eukaryotes – the centromere is composed of hundreds of thousands of base pairs consisting of tandem repeats of short sequences; it is unclear how these sequences provide specificity to the centromere o Specific chromatin structure may be involved in this specificity (eg/ a specific Histone H3 variant is only found in the chromatin of centromeres) 10 BIO 2C03 2013  Telomere o Found at the ends of chromosomes o Function as a “cap” protecting and stabilizing the chromosomes o The loss of telomeres generates sticky chromosomes that tend to stick to each other and are more susceptible to degradation o Telomeres also provide the means of replicating the ends of chromosomes o Telomeres are dynamic structures that may function as an “internal clock” determining the number of cell divisions and the replication potential of the normal cell o Telomere shortening results in “senescence” i.e. the inability of the cell to proliferate (“irreversible growth arrest”) o Cancer cells often escape senescence o Structure consist of repeated sequences which are generally a series of cytosine nucleotides followed by A-T rich sequence o In humans – the CCCTAA sequence may be repeated 250 to 1500 times o The cytosine nucleotides are located at the 5’ end of the DNA strand i.e. toward the end of the chromosome o The complementary strand (“G-rich” strand) protrudes as single-stranded DNA o The G-rich strand can also invade the double-stranded region to form a “t-loop” structure that protects the end of the chromosome o The telomere is also protected by a protein complex called the “Shelterin Complex” which includes both double-stranded DNA (TRF1 and TRF2) and single-stranded DNA binding proteins (POT1) o In Drosophila – telomeres consist of multiple copies of two different transposable elements called Het-A and Tart, organized in tandem repeats o Adjoining the telomeric repeats are “telomere-associated sequences” consisting of several thousands to hundred of thousands of base pairs o The telomere-associated sequences are also composed of repeats that are longer, more complex are more varied in sequence than the telomeric repeats (true in all organisms)  Artificial Chromosome o It is now possible to generate artificial chromosomes that are stable, replicated and segregated faithfully o Required elements:  Origin of replication  Centromeres  Telomeres 11 BIO 2C03 2013 o Eg/ YAC (Yeast A. T.) o Eg/ MAC (Mammalian A. C.) o Eg/ BAC (Bacterial A.C.) – counterpart in prokaryotes o BAC and YAC played a critical role in the sequencing of the human genome Single Copy DNA vs. Repetitive DNA  Genomic sequences can also be classified according to the number of copies or repeats  Three different classes of DNA sequences are generally recognized: 1. Single Copy DNA  Include most protein coding genes  Found as a single copy per haploid genome or as a small gene family of highly related sequences (eg/ 7 β-globin genes clustered on chromosome 11 2. Moderately Repetitive DNA Sequences  Some consist of a few hundred copies (eg/ rRNA genes, tRNA genes)  Some are found as tandem repeat sequences (located in a discrete region of the genome)  Others are found as interspersed repeat sequences and thus are dispersed throughout the genome  Often have no known functions  Interspersed repeat sequences are generally transposable elements that can replicate and move (“mobile elements”)  Interspersed repeat sequences can be classified according to size a) Short Interspersed Elements (SINEs) – eg/ “Alu” sequence is 200 bp long and is found as a million copies scattered throughout the human genome b) Long Interspersed Elements (LINEs) – eg/ “L1 element” is 6000-8000 bp in length; there is about 100,000 copies of L1 representing about 15% of the human genome 3. Highly Repetitive DNA Sequences:  Short sequences (10 bp) found as hundreds of thousands or millions of copies, repeated in tandem and clustered in certain regions of the chromosome (eg/ near the centromere and telomeres)  Known as “satellite DNA” because they migrated as a separate DNA fraction following centrifugation at high speed due to their different base composition  No clear function has been assigned to highly repetitive DNA sequences  Satellite DNA – A-T rich and has lower density than that of the bulk of the DNA; can be isolated from fragmented DNA using ultracentrifugation of density gradient (eg/ CaCl gradient) 12 BIO 2C03 2013 Organization of the genome Lecture 3 – DNA Replication (March 1) Chapter 12 Introduction  DNA Polymerases must have high processivity o Eg/ in optimal conditions, E. coli can divide every 20 minutes o DNA replication proceeds at a rate of 1000 n. per second  DNA polymerases must be accurate o Eg/ in E. coil, the error rate is one nucleotide per billion  DNA synthesis is initiated at the “origin of replication” and proceeds through the entire “replicon” or unit of replication  The E. coli chromosome represents a single replicon  Eukaryotic DNA contains several origins of replication and replicons DNA Synthesis is Semiconservative  Three proposed mechanisms A. Conservative B. Dispersive C. Semiconservative  Messelson and Stahl, 1958 13 BIO 2C03 2013 Mechanisms of Replication 1) Theta Replication of circular DNA (eg/ E. coli) o DNA unwinding generates a “replication bubble” o The point of unwinding is known as the “replication fork” o DNA replication proceeds in a 5’ to 3’ direction on both strands 2) Rolling-Circle Replication – circular DNA in F Factor and some viruses 3) Linear Eukaryotic DNA Replication o Replication is slower in eukaryotes (500-5000 nucleotides permin) o There are more origins of replication o Each replicon is 20,000 to 300,000 nucleotides in length 14 BIO 2C03 2013  Basic Reaction  Leading vs. Lagging Strand  Okazaki Fragment – DNA fragments generated by discontinuous replication; 1000-2000 nucleotides long in prokaryotes  There are no replication bubbles in molecules replicated by the rolling circle mechanism 15 BIO 2C03 2013  In eukaryotes – the Okazaki fragments are 100-200 nucleotides long Proteins Involved in DNA Replication 1) Prokaryotes o Important rules in DNA Replication i. Replication is only initiated at specific “origins” (eg/ oriC in E. coli) ii. Replication proceeds in a 5’ to 3’ direction iii. To initiate replication, DNA Polymerases need a primer to provide a 3’ –OH group and begin the polymerization reaction i.e. they cannot begin replication on a bare template (unlike RNA polymerases) iv. Primers are small RNAs synthesized by specialized enzymes (DNA primases) v. In addition to DNA Polymerases, several enzymes and factors are required to unwind the DNA, prevent the formation of secondary structure, relieve the supercoiling and seal the gaps between Okazaki fragments o Overview o Replication requires the synthesis of RNA primers by primases  The leading strand requires a single primer while multiple primers are required for synthesis of Okazaki fragments on the lagging strand  Primers are 10 nucleotides long  The primase forms a complex with the helicase at the replication fork (coupling) o DNA Pol III replaces the ribonucleotides of the primer by deoxy-ribonucleotides o A phosphodiester bond, catalyzed by DNA ligase, seals the nick between adjacent DNA fragments 16 BIO 2C03 2013 o Several Polymerases and DNA ligase are required for replication o DNA replication n prokaryotes is fast (1000 nucleotides/sec) and accurate (1 error/100000 nucleotides inserted) o Proofreading by DNA Polymerases further reduces the error rate to 1 nucleotide per 10,000,000 nucleotides o DNA mismatch repair can also eliminate mismatched nucleotides o DNA Polymerases also have 3’-5’ exonuclease activity, endowing them with proofreading capability o Incorporation of the wrong nucleotide in the growing DNA chain causes DNA Pol to “stall” – stalls because the 3’ OH group required for the addition of the incoming nucleotide is not positioned properly in the catalytic site  DNA is distorted by the addition of the incorrect nucleotide o Stalling of DNA Pol leads to removal of the incorrectly positioned nucleotide by the 3’ to 5’ exonuclease activity of the enzyme o DNA Pol resumes synthesis and proceeds with the incorporation of the correct nucleotide 2) Eukaryotes o Origins of Replication and its Protein Complex  Origins of Replication were first isolated in yeast and shown to function as “autonomously replicating sequences” (ARS)  The sequence of origins varies considerably among eukaryotes but are generally A-T rich sequences of 100-200 bp in length  Origins are recognized by a multiprotein complex, designed ORC (origin-recognition complex)  The ORC binds to the origins and unwinds the DNA, thus providing the conditions to initiate replication o Licensing of DNA replication (timing is everything)  The existence of multiple origins of replication could lead to under- or over-replication  Specific mechanisms exist to ensure that all DNA is replicated only once per cell cycle  For replication to proceed the origin must fist be recognized (i.e. bound to) by a “replication licensing factor” which recruits the replication machinery  Progression of the replication form leads o the removal of the licensing factor and return to of the origin to an “unlicensed status”  The licensing factor is known as the “minichromosome maintenance” complex or MCM  After initiation, the MCM complex is sequestered by a protein called Geminin ensuring that re-initiation does not take place  Sequestration ends when Geminin is degraded at the end of mitosis, enabling the MCM complex to bind to the origins of replication and re-initiate DNA synthesis o Eukaryotic cells express several DNA polymerases 17 BIO 2C03 2013 o High fidelity vs low fidelity DNA Polymerases  Distorted DNA templates, resulting from DNA damage, will impede (stall) the progression of high fidelity DNA polymerases such as DNA Pol. Ε or δ  To bypass these lesions, cells also express “low fidelity” DNA polymerases that can use a distorted template for DNA synthesis  These DNA Pol (eg/ ζ, ι, κ) are involved in “translesion DNA synthesis”  DNA repair systems detect and correct these lesions (most of the time)  High fidelity DNA polymerases resume synthesis beyond the DNA translesion, resuming synthesis with great accuracy o Nucleosomes are quickly assembled on newly synthesized DNA Lecture 4 – Transcription (March 5) Chapter 13 The mRNA Hypothesis  Proteins are made on ribosomes using information encoded in DNA  In eukaryotes, ribosomes are in the cytoplasm but most of the DNA is in the nucleus  Therefore, how is genetic information transferred from the nucleus to the cytoplasm?  What is the nature of the “messenger”?  Proteins are made on ribonucleotproteins i.e. ribosomes  Therefore, is the messenger RNA? Is it ribosomal RNA or another RNA species  Is RNA made afterphage infection; if so what kind of RNA is made after infection (Hershey Chase) o Observed – infection of E.coli by Phage T2 causes the appearance of a new class of rare RNA (circa 1-2% of total RNA) o FOR RNA (F. Crick) – rRNA is abundant, is on ribosomes o AGAINST RNA (Jacob and Monod) – rRNA is homogenous in size (but proteins are not), is abundant (too abundant) stable  Brenner, Jacob, Meselson, Gros: E. coli infection by phage T4 1. Pre-label E. coli ribosomes with heavy N and C isotopes 2. Add P and S to label nucleic acids and proteins, respectively 3. Infect E. coli with phage T4 The observed 1. All newly synthesized RNA is associated with heavy ribosomes 2. All newly synthesized RNA is heterogeneous in size and is not rRNA 3. All newly synthesized proteinsare associated with heavy ribosomes 4. No light ribosomes are synthesized after infection 18 BIO 2C03 2013 5. Therefore, the messenger is not ribosome and rRNA but a different class of rare, heterogeneous RNA i.e. mRNA  The mRNA hypothesis o The messenger should be (Monod, Jacob et al; 1961) – heterogeneous in size; transiently associated with the ribosome; rare; unstable o Spiegelman then confirms through RNA-DNA hybridization that the newly synthesized RNA is complementary to phage DNA and not E. coli DNA o Therefore – the heterogeneous RNA is the messenger encoded by phage DNA and translated into phage proteins on E. coli ribosomes Structure of RNA  Uracil replaces thymine in RNA  The sugar is ribose (with its 2’ OH group)  The presence of the 2’OH makes the RNA more susceptible to degradation by alkali and attach by heavy metals  The RNA is a single stranded polynucleotide chain that forms varied and extensive secondary structure  However, extensive regions of the RNA exist as “single stranded loops” that are more accessible to endonucleases and degradation  RNA generally exists as a ribonucleoprote
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