Nucleic Acids: DNA to DNA & DNA to RNA PDF

Case 2a - Nucleic acids: from DNA to DNA and DNA to RNA 1. What is the function of DNA? Deoxyribonucleic acid (DNA) is a molecule that contains the biological instructions that make each species unique. DNA, along with the instructions it contains, is passed from adult organisms to their offspring...

Case 2a - Nucleic acids: from DNA to DNA and DNA to RNA 1. What is the function of DNA? Deoxyribonucleic acid (DNA) is a molecule that contains the biological instructions that make each species unique. DNA, along with the instructions it contains, is passed from adult organisms to their offspring during reproduction. - DNA contains instructions needed to develop, survive and reproduce - DNA → converted in messages → produce proteins - DNA sequence = gene (1,000 bases - 1 million bases) (1). 2. What is the structure of DNA? The functional unit of genetic information is the gene, defined as a length of DNA that directs the synthesis of a polypeptide or of a functional ribonucleic acid (RNA). What are the bases? - Four bases - Polymer of nucleoside monophosphates - Back bone → alternating phosphate and 2-deoxyribose → phosphodiester bonds - Carbon 3 and 5 of sugar is used for the phosphodiester bonds - Carbon 1 forms a β- N -glycosidic bond with 1 base - One end free hydroxyl group → C-5 (5’) - Other end at C-3 (3’) - 5’-terminus written left, 3’-terminus written right; 5’ → 3’  - The purines (2 circles) in DNA are adenine and guanine, the same as in RNA. The pyrimidines (1 circle) in DNA are cytosine and thymine; in RNA, they are cytosine and uracil. What is a double helix? Cellular DNA → double strand, double helix - The strands run in opposite direction - Backbone → two ridges on surface molecule - Phosphate group = negative charge - Bases face inward but edges exposed - Bases form the minor and major groove - Bases lie flat on top of each other, flat surfaces = hydrophobic, successive bases in strand form van der waals verbindingen - A - T → 2 hydrogen bonds - G - C → 3 hydrogen bonds - The double strand is wound into a right-handed helix. Each turn of the helix has about 10.4 base pairs and advances about 3.4 nm along the helix axis. The double helix is rather stiff, but it can be bent and twisted by DNA-binding proteins. What is supercoiling? Supercoiling reduces space and more packaging. In eukaryotes, DNA supercoiling exists on many levels of both plectonemic and solenoidal supercoils, with the solenoidal supercoiling proving the most effective in compacting the DNA. Solenoidal supercoiling is achieved with histones (a protein that provides structural support for a chromosome) to form a 10 nm fiber. This fiber is further coiled into a 30 nm fiber, and further coiled upon itself numerous times more (2). - Nucleosomes contain 8 histones → octamer - H2A and H2B are one of the main proteins found in histones - H2A, H2B, H3 and H4 → core proteins of histones. Core formation first occurs through the interaction of two H2A molecules. Then, H2A forms a dimer with H2B; - The DNA twirls twice around histones - Nucleosome: 10 nm - Chromatin: 700 nm - H1 keeps the wrapped DNA around the core histone in place - Solenoid → nucleosome wrapped around each other Non-histone proteins: further compaction Eukaryotes Many naturally occurring DNA molecules are circular. When a linear duplex is partially unwound by one or several turns before it is linked into a circle, the number of base pairs per turn of the helix is greater than the usual 10.4. The torsional strain in this molecule leads to supercoiling of the duplex around its own axis, much as a telephone cord twists around itself. This is called a negative supertwist. The opposite situation, in which the helix is overwound, is called a positive supertwist. - Most cellular DNA’s → negative twist → 5 to 7 % less right hand turns → underwound condition → helps unwinding of DNA - Topoisomerase → regulates supertwisting The supertwisting of DNA is regulated by two types of topoisomerase. Type I topoisomerases cleave one strand of the double helix, creating a molecular swivel that relaxes supertwists passively. Type II topoisomerases are more complex. They cleave both strands and allow an intact helix to pass through this transient double- strand break, before resealing the break. Type II topoisomerases relax positive supertwists passively and use ATP hydrolysis to pump negative supertwists into the DNA. Prokaryotes In prokaryotes, plectonemic supercoils are predominant, because of the circular chromosome and relatively small amount of genetic material. - No nucleus, only coding DNA, circular DNA Differences pro and eukaryotes Property Prokaryotes Eukaryotes Typical size 0.4–4 μm 5–50 μm Nucleus – + Membrane-bounded organelles – + Cytoskeleton – + Endocytosis and exocytosis – + Cell wall + (some –) + (plants) – (animals) No. of chromosomes 1 (+ plasmids) >1 Ploidy Haploïd Haploid or diploid Histones – + Introns – + Ribosomes 70S 80S - Silencers are regulatory DNA elements that reduce transcription from their target promoters; they are the repressive counterparts of enhancers. - A TATA box is a DNA sequence that indicates where a genetic sequence can be read and decoded. It is a type of promoter sequence, which specifies to other molecules where transcription begins. - A codon is a DNA or RNA sequence of three nucleotides (a trinucleotide) that forms a unit of genomic information encoding a particular amino acid or signaling the termination of protein synthesis (stop signals). There are 64 different codons: 61 specify amino acids and 3 are used as stop signals. Mitochondrial DNA - 1000 mitochondria per cell - 5-10 mitochondrial genomes per mitochondria - Tiny genome: 17 kb (kilobases) - Genes: - 2 rRNA’s - 22 tRNA’s - 13 proteins which are subunits of: NADH dehydrogenase, cytochrome oxidase, ATP synthase, cytochrome b 3. How is DNA replicated? 3 major steps: 1. Opening of double helix + separation of DNA strands → requires ATP-dependent enzymes 2. Priming of template strand 3. Assembly of new DNA segment Separation → strands uncoil at origin Several enzymes + proteins work together to prime DNA polymerase organizes assembly. What triggers replication? Initiation 2 steps: 1. Initiator proteins unwinds short stretch of double helix When DNA needs to be replicated → mRNA that codes for initiation proteins will be translated → bind to origins of replication, bind to AT rich sequences → easier to break hydrogen bonds 2. Helicase → attaches to and breaks apart hydrogen bonds While moving along the strand helicase breaks apart → separating the polynucleotide chain AT rich region will open up → binding of single stranded binding proteins + helicase Helicase will open up structure both sides → SSB will prevent hydrogen bonds to form again DNA unwinding creates the replication fork. 17 base pairs for the replication bubble. Meanwhile DNA primase briefly attaches to each strand → assembles foundation where replication can begin → short stretch of nucleotides = RNA primer → DNA pol can only add the free nucleotides to a free 3’ site end. Primer is between 2-5 nucleotides. (10 prokaryotes) How are DNA strands replicated? - After primer is placed → DNA polymerase wraps itself around that strand → attaches new nucleotides to exposed nitrogenous bases - DNA polymerase assembles new strand on top of existing one in the 5′ → 3′ direction, while reading the template in the 3′ → 5′ direction. - Needs free nucleotides to assemble new strand - Base pairing → strands match - Complementary strand → anti sequence of template strand DNA polymerase have exonuclease activities (prokaryotes) In prokaryotes, three main types of polymerases are known: DNA pol I, DNA pol II, and DNA pol III. - DNA pol I: fills gaps left by removal of RNA primer (5’3’ exonuclease activity) and involved in DNA repair. - DNA pol II: involved in DNA repair DNA pol III The major enzyme of DNA replication in E. coli is DNA polymerase III ( poly III ), a very fast enzyme that polymerizes up to 1000 nucleotides per second. It also has very high processivity. This means it binds very tightly to the template strand. It does not fall off the template until the entire bacterial chromosome has been replicated. Challenge in replication → accuracy → if not then mutation Minimize this → poly III has a 3′-exonuclease activity → used for proofreading Nucleases cleave phosphodiester bonds in nucleic acid Deoxyribonucleases (DNases) cleave DNA, and ribonucleases (RNases) cleave RNA. Nucleases that cleave internal phosphodiester bonds are called endonucleases Those that remove nucleotides from the 5′ end or the 3′ end are called exonucleases. 3’-exonuclease activity of poly III comes into play only when the nucleotide that has been added to the 3′ end of a growing chain fails to pair with the base in the template strand → the last nucleotide is removed. Proofreading mechanism reduces the error rate from 1 in 10^4 or 1 in 10^5 to less than 1 in 10^7. - Free base has 3 phosphate groups → removing two phosphate groups creates energy needed to make the phosphodiester bond  - DNA pol III has a P site and an E site, P: polymerase site, E: exonuclease - Wrong base → structural change → strand is folded to E site and edited One of the new DNA strands is synthesized discontinuously DNA polymerases synthesize only in the 5′ → 3′ direction, reading their template 3′ → 5′. Because the parental double strand is antiparallel, only one of the new DNA chains, the leading strand, can be synthesized by a poly III molecule that travels with the replication fork. The other strand, called the lagging strand, has to be synthesized piece by piece. This requires the repeated action of the primase, followed by poly III. Together they produce DNA strands of about 1000-2000 nucleotides, each with a little piece of RNA at the 5′ end. These strands are called Okazaki fragments. The RNA primer is soon removed by the 5′-exonuclease activity of poly I. The gaps are filled by its polymerase activity, but poly I cannot connect the loose ends of two Okazaki fragments. This is the task of a DNA ligase, which links the phosphorylated 5′ terminus of one fragment with the free 3′ terminus of another. Strangely, bacteria derive the energy for this reaction from the hydrolysis of the phosphoanhydride bond in NAD, a coenzyme that is otherwise used for hydrogen transfers, although humans use ATP. Role of supercoiling Supercoiling plays a role in front of the helicase. The two strands in the replication fork are opened up, this causes the DNA to supercoil in front of the helicase. The amount of turns are present in a smaller area, this causes positive supercoiling. This makes it difficult for the helicase to move. Topoisomerases will remove this supercoiling, by cleaving, to help the helicase move along the DNA strand. Eukaryotic Generally similar to prokaryotic: - Semi conservative - Bidirectional - Semi discontinuous  - Requires primer Except: - Larger genomes require multiple origins - Usually more enzymes involved (more complex >200 proteins involved) - Names of enzymes might be different - Okazaki fragments are much shorter - Need to deal with nucleosomes - Instead of a primer of 2-5 nucleotides → 5-8 nucleotides - Okazaki fragments of 100-200 nucleotides DNA polymerases involved Eukaryotic cells contain five DNA polymerases: α, β, γ, δ, and ε. Polymerase γ is located in mitochondria and is responsible for replication of mitochondrial DNA. The other four enzymes are located in the nucleus and are therefore candidates for involvement in nuclear DNA replication. Polymerases α, δ, and ε are most active in dividing cells, suggesting that they function in replication. In contrast, polymerase β is active in nondividing and dividing cells, suggesting that it may function primarily in the repair of DNA damage. - Exonuclease activity is missing, RNAses remove the primers Sliding clamp The polymerase can fall off the DNA, can not attach very easily to DNA. There are additional proteins forming a sliding clamp, it wraps around the DNA and the polymerase binds to the sliding clamp and therefore the polymerase can not fall off the DNA.