L8 2025 - DNA PDF

 How the information carried in DNA is used to specify the structures of protein products. Lectures 8,9,10 DNA, RNA, Proteins Professor George Baillie ([email protected]) INSTITUTE OF CARDIOVASCULAR AND MEDICAL SCIENCES TWITTER: Baillie Lab@baillie_lab Biology 1B...

 How the information carried in DNA is used to specify the structures of protein products. Lectures 8,9,10 DNA, RNA, Proteins Professor George Baillie ([email protected]) INSTITUTE OF CARDIOVASCULAR AND MEDICAL SCIENCES TWITTER: Baillie Lab@baillie_lab Biology 1B Semester 2, (23rd, 27th, 28th Jan 2025) Lecture 8 DNA: a store of biological information Lecture 9 RNA: how genes are expressed Lecture 10 : How cells make proteins  Learning Objectives Lecture 8 (DNA) Describe evidence that DNA carries genetic information Show that DNA is an anti-parallel double-stranded polynucleotide in which base-pairing plays a crucial role Explain the Meselson-Stahl experiment to show semi-conservative replication of DNA Show how DNA polymerase and other proteins achieve DNA replication Explain that DNA replication is a very accurate process but that rare mistakes can lead to mutations  Scientific inspiration. The origins of protein phosphorylation, Philip Cohen, 2002, NCB, 4, E127-E130 Carl and Gerty Cori 1947 (glycogen metabolism) Earl Sutherland 1971 (cAMP) Crebs and Fischer 1992 (PKA) Gilman and Rodbel 1994 (G proteins) Science. 2002 Oct 25;298(5594):834-6. Targeting of cyclic AMP Lefkowitz/Kobilka 2012 degradation to beta 2-adrenergic receptors by beta-arrestins. (G protein couled receptors) Baillie GS, Perry, S and Lefkowitz  (1844-1895) (1822-1884) (1866-1945) 1879-1941 Transformation is a change in genotype & phenotype due to assimilation of external DNA by a cell…but at the time of Griffith the identity of the “inheritance factor” remained a mystery though widely thought to be protein.  Avery carried on Griffiths work and systematically destroyed lipids, carbohydrate, proteins and ribonucleic acid of virulent bacteria BUT transformation still occurred. Only when he used deoxyribonuclease to destroy DNA was transformation blocked. This was the first indication that DNA was the inheritance factor. Oswald T. Avery 1877-1937 Other evidence came from studies of viruses which infect bacteria (phages). Only the DNA (32P label), not the protein (35S label) enters the bacterial cell. T2 Alfred Hershey This was final proof that DNA was genetic material, now race was on to determine how structure of DNA could account for its role in inheritance Martha Chase The composition of DNA Erwin Chargaff  The composition of DNA In 1947, DNA was known to be a polymer of nucleotides (base-sugar (deoxyribose)-phosphate). Though nothing known of its structure. Each nucleotide consists of a base (T,A,CorG), the sugar deoxyribose (blue) and a phosphate (yellow)  The structure of DNA From X-ray diffraction experiments (Wilkins and Franklin, early 1950s), it was known that DNA was helical in shape. Rosalind Franklin 1920 - 1958 “Picture 51” 1952 This allowed Watson to deduce width of helix and spacing of nitrogenous bases along it.  The structure of DNA * Watson and Crick  The structure of DNA-Features of the Watson and Crick model 1. Sugar-phosphate backbone 2. The strands run anti-parallel to each other (5’-3’ and 3’-5’) 3. 10 base pairs per turn of the double helix 4. The bases (non-polar hydrophobic) are on inside, the phosphate (polar, hydrophilic) are on outside. 5. Space filling models show a major and a minor groove in the double helix Replication of DNA The Watson-Crick model suggested a mechanism for copying the genetic information from one cell to its daughter cells. Each strand acts as the template for synthesis of a new stand according to the base-pairing rules. The two strands need to unwind and separate to expose the bases to allow the synthesis of new DNA to occur.  Replication of DNA This type of mechanism is termed "semi- conservative", as opposed to "conservative" or "dispersive" models. Evidence for the semi-conservative model came from an elegant experiment carried out by Meselson and Stahl (1958). Stahl 1929- Meselson 1930-  Replication of DNA The experiment relied on:- Escherichia coli, a bacterium which could grow on simple sources of N and C isotopes of N (15N and 14N) to distinguish between old and new DNA density gradient centrifugation to separate the two types (15N-DNA is about 1% more dense than 14N-DNA) The results were only consistent with the semi- conservative model. DNA replication is elegantly simple… BUT the biochemistry is complex. *  Replication of DNA Many proteins are involved in the replication of DNA Replication starts at specific origins of replication (one in prokaryotes, many in eukaryotes). Proteins recognise the DNA sequence and bind to it, opening up the double helix. In the replication bubble, there are Y-shaped replication forks, where the new strands are being elongated. Replication starts at multiple sites where the parental strands separate to form replication bubbles. The bubbles expand laterally as DNA replication proceeds in both directions Eventually the replication bubbles fuse and synthesis of the daughter strands is complete  Replication of DNA The elongation is carried out by enzymes known as DNA polymerases. They use triphosphates (ATP, GTP etc.) as the sources of nucleotide units to add to the growing chains. A two-phosphate unit (pyrophosphate) is split out as the chain is extended by each nucleotide. Rate of elongation is 50 nucleotides a second in humans. Now over 10 different DNA polymerases identified in eukaryotes. Note that nucleotides can ONLY be added to the free 3’ end..NEVER the 5’end.  Replication of DNA Because the strands are anti-parallel and the enzymes can extend chains in one direction only (5´ 3´), the 2 strands cannot be both replicated continuously. We thus have a leading strand (continuous replication) and a lagging strand (discontinuous replication). * Replication of DNA The fragments of the lagging strand (Okazaki fragments) are typically 100-200 nucleotides in length and are later joined by an enzyme (DNA ligase). DNA polymerases can only extend a pre- existing strand - so how does the process start? The answer is that the cell starts by making a short primer of RNA (RiboNucleicAcid), about 10 nucleotides long, which is added to. This primer is later replaced by DNA. Replication of DNA is an extremely rapid and accurate process. Summary of important proteins in DNA replication Lecture 8 summary slide Familiarise yourself with the ground breaking discoveries of Mendel, Miesher, Morgan, Griffith, Avery, Hershey/Chase, Chargaff, Meselson/Stahl, Watson/Crick Know that the DNA polymerase mechanism on the leading/lagging strand during DNA replication is different. Remember the names and roles of other proteins in the process of DNA replication.  How the information carried in DNA is used to specify the structures of protein products. Lectures 8, 9, 10 DNA, RNA, Proteins Professor George Baillie ([email protected]) INSTITUTE OF CARDIOVASCULAR AND MEDICAL SCIENCES TWITTER: Baillie Lab@baillie_lab Biology 1B Semester 2, (23, 27, 28 Jan 2025) Lecture 8 DNA: a store of biological information Lecture 9 RNA: how genes are expressed Lecture 10 : How cells make proteins Learning Objectives Lecture 8 (DNA) Describe evidence that DNA carries genetic information Show that DNA is an anti-parallel double-stranded polynucleotide in which base-pairing plays a crucial role Explain the Meselson-Stahl experiment to show semi- conservative replication of DNA Show how DNA polymerase and other proteins achieve DNA replication Explain that DNA replication is a very accurate process but that rare mistakes can lead to mutations  Scientific inspiration. The origins of protein phosphorylation, Philip Cohen, 2002, NCB, 4, E127-E130 Carl and Gerty Cori 1947 (glycogen metabolism) Earl Sutherland 1971 (cAMP) Crebs and Fischer 1992 (PKA) Gilman and Rodbel 1994 (G proteins) Science. 2002 Oct 25;298(5594):834-6. Targeting of cyclic AMP Lefkowitz/Kobilka 2012 degradation to beta 2-adrenergic receptors by beta-arrestins. (G protein couled receptors) Baillie GS, Perry, S and Lefkowitz  (1844-1895) (1822-1884) (1866-1945) 1879-1941 Transformation is a change in genotype & phenotype due to assimilation of external DNA by a cell…but at the time of Griffith the identity of the “inheritance factor” remained a mystery though widely thought to be protein.  Avery carried on Griffiths work and systematically destroyed lipids, carbohydrate, proteins and ribonucleic acid of virulent bacteria BUT transformation still occurred. Only when he used deoxyribonuclease to destroy DNA was transformation blocked. This was the first indication that DNA was the inheritance factor. Oswald T. Avery 1877-1937 Other evidence came from studies of viruses which infect bacteria (phages). Only the DNA (32P label), not the protein (35S label) enters the bacterial cell. T2 Alfred Hershey This was final proof that DNA was genetic material, now race was on to determine how structure of DNA could account for its role in inheritance Martha Chase The composition of DNA Erwin Chargaff The composition of DNA In 1947, DNA was known to be a polymer of nucleotides (base-sugar (deoxyribose)-phosphate). Though nothing known of its structure. Each nucleotide consists of a base (T,A,CorG), the sugar deoxyribose (blue) and a phosphate (yellow) TheFrom structure of DNA X-ray diffraction experiments (Wilkins and Franklin, early 1950s), it was known that DNA was helical in shape. Rosalind Franklin 1920 - 1958 “Picture 51” 1952 This allowed Watson to deduce width of helix and spacing of nitrogenous bases along it. The structure of DNA * Watson and Crick The structure of DNA-Features of the Watson and Crick model 1. Sugar-phosphate backbone 2. The strands run anti-parallel to each other (5’-3’ and 3’-5’) 3. 10 base pairs per turn of the double helix 4. The bases (non-polar hydrophobic) are on inside, the phosphate (polar, hydrophilic) are on outside. 5. Space filling models show a major and a minor groove in the double helix Replication of DNA The Watson-Crick model suggested a mechanism for copying the genetic information from one cell to its daughter cells. Each strand acts as the template for synthesis of a new stand according to the base-pairing rules. The two strands need to unwind and separate to expose the bases to allow the synthesis of new DNA to occur. Replication of DNA This type of mechanism is termed "semi- conservative", as opposed to "conservative" or "dispersive" models. Evidence for the semi-conservative model came from an elegant experiment carried out by Meselson and Stahl (1958). Stahl 1929- Meselson 1930- Replication of DNA The experiment relied on:- Escherichia coli, a bacterium which could grow on simple sources of N and C isotopes of N (15N and 14N) to distinguish between old and new DNA density gradient centrifugation to separate the two types (15N-DNA is about 1% more dense than 14N-DNA) The results were only consistent with the semi- conservative model. DNA replication is elegantly simple… BUT the biochemistry is complex. * Replication of DNA Many proteins are involved in the replication of DNA Replication starts at specific origins of replication (one in prokaryotes, many in eukaryotes). Proteins recognise the DNA sequence and bind to it, opening up the double helix. In the replication bubble, there are Y-shaped replication forks, where the new strands are being elongated. Replication starts at multiple sites where the parental strands separate to form replication bubbles. The bubbles expand laterally as DNA replication proceeds in both directions Eventually the replication bubbles fuse and synthesis of the daughter strands is complete Replication of DNA The elongation is carried out by enzymes known as DNA polymerases. They use triphosphates (ATP, GTP etc.) as the sources of nucleotide units to add to the growing chains. A two-phosphate unit (pyrophosphate) is split out as the chain is extended by each nucleotide. Rate of elongation is 50 nucleotides a second in humans. Now over 10 different DNA polymerases identified in eukaryotes. Note that nucleotides can ONLY be added to the free 3’ end..NEVER the 5’end. Replication of DNA Because the strands are anti-parallel and the enzymes can extend chains in one direction only (5´ 3´), the 2 strands cannot be both replicated continuously. We thus have a leading strand (continuous replication) and a lagging strand (discontinuous replication). * Replication of DNA The fragments of the lagging strand (Okazaki fragments) are typically 100-200 nucleotides in length and are later joined by an enzyme (DNA ligase). DNA polymerases can only extend a pre- existing strand - so how does the process start? The answer is that the cell starts by making a short primer of RNA (RiboNucleicAcid), about 10 nucleotides long, which is added to. This primer is later replaced by DNA. Replication of DNA is an extremely rapid and accurate process. Summary of important proteins in DNA replication Lecture 13 summary slide Familiarise yourself with the ground breaking discoveries of Mendel, Miesher, Morgan, Griffith, Avery, Hershey/Chase, Chargaff, Meselson/Stahl, Watson/Crick Know that the DNA polymerase mechanism on the leading/lagging strand during DNA replication is different. Remember the names and roles of other proteins in the process of DNA replication.