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DNA and Genetic Material Chapters 3 and 25 Yes, we are moving around a little! • Composed of 4 nucleotide bases, 5 carbon sugar and phosphate. • Base pair = rungs of a ladder. • Edges = sugar-phosphate backbone. • Double Helix • Anti-Parallel The bases • Chargaff’s Rules • A=T • G=C • led to suggestion of a double helix structure for DNA The Bases • Adenine (A) always base pairs with thymine (T) • Guanine (G) always base pairs with Cytosine (C) The Bases • The C#T pairing on the left suffers from carbonyl dipole repulsion, as well as steric crowding of the oxygens. The G#A pairing on the right is also destabilized by steric crowding (circled hydrogens). DNA Replication • • • • Adenine (A) always base pairs with thymine (T) Guanine (G) always base pairs with Cytosine (C) ALL Down to HYDROGEN Bonding Requires steps: – H bonds break as enzymes unwind molecule – New nucleotides (always in nucleus) fit into place beside old strand in a process called Complementary Base Pairing. – New nucleotides joined together by enzyme called DNA Polymerase DNA Replication • Each new double helix is composed of an old (parental) strand and a new (daughter) strand. • As each strand acts as a template, process is called Semi-conservative Replication. • Replication errors can occur. Cell has repair enzymes that usually fix problem. An error that persists is a mutation. • This is permanent, and alters the phenotype. DNA replication •If only is was that simple………. DNA replication • • • • • • Origins of replication The replication fork Leading strand Lagging strand Dynamics at the replication fork Regulation of replication Origins of replication • Targeted by proteins that separate the two strands and initiate DNA synthesis. • Contain DNA sequences recognized by replication initiator proteins • These initiator proteins recruit other proteins to separate the two strands and initiate replication forks Origins of replication • Initiator proteins recruit other proteins to separate the DNA strands at the origin, forming a bubble. • Tend to be "AT-rich" to assist this process • A-T base pairs have two hydrogen bonds strands rich in these nucleotides are generally easier to separate due the positive relationship between the number of hydrogen bonds and the difficulty of breaking these bonds. The replication fork • structure that forms within the nucleus during DNA replication • created by helicases : break H bonds between bases. The resulting structure has two branching "prongs", each one made up of a single strand of DNA. Leading & Lagging strands • The leading strand template is oriented in a 3' to 5' manner. • All DNA synthesis occurs 5'-3'. The original DNA strand must be read 3'-5' to produce a 5'-3' daughter strand. Leading & Lagging strands • The lagging strand template is the coding strand of the DNA double helix that is oriented in a 5' to 3' manner . • The newly made lagging strand still is synthesized 5'-3'. • WHAT? Leading & Lagging strands • The DNA is oriented in a manner that does not allow continual synthesis, only small sections can be read at a time. • An RNA primer is placed on the DNA strand 3' to the origin of replication. Just as before, DNA Polymerase reads 3'-5' on the original DNA to produce a 5'-3' daughter strand. • Polymerase reaches the origin of replication and stops replication until a new RNA primer is placed 3' to the last RNA primer. Okazaki fragments • Fragments of DNA produced on the lagging strand. • The orientation of the original DNA on the lagging strand prevents continual synthesis. As a result, replication of the lagging strand is more complicated than of the leading strand. • In eukaryotes, primase is intrinsic to DNA polymerase III which also lengthens the primed segments, forming Okazaki fragments. Primer removal in eukaryotes is also performed by this enzyme. Dynamics at the replication fork • As helicase unwinds DNA at the replication fork, the DNA ahead is forced to rotate. • Results in a build-up of twists in the DNA ahead. This buildup would form a resistance that would eventually halt the progress of the replication fork. • DNA topoisomerases are enzymes that solve these physical problems in the coiling of DNA. • Topoisomerase I cuts a single backbone on the DNA, enabling the strands to swivel around each other to remove the build-up of twists. Dynamics at the replication fork • Topoisomerase II cuts both backbones, enabling one double-stranded DNA to pass through another, thereby removing knots and entanglements that can form within and between DNA molecules. • Bare single-stranded DNA has a tendency to fold back upon itself and form secondary structures; these structures can interfere with the movement of DNA polymerase. • To prevent this, single-strand binding proteins bind to the DNA until a second strand is synthesized, preventing secondary structure formation Summary • Topoisomerase: This enzyme initiates unwinding of the double helix by cutting one of the strands. • Helicase: This enzyme assists the unwinding. Note that many hydrogen bonds must be broken if the strands are to be separated.. • SSB: A single-strand binding-protein stabilizes the separated strands, and prevents them from recombining, so that the polymerization chemistry can function on the individual strands. • DNA Polymerase: This family of enzymes link together nucleotide triphosphate monomers as they hydrogen bond to complementary bases. These enzymes also check for errors (roughly ten per billion), and make corrections. • Ligase: Small unattached DNA segments on a strand are united by this enzyme. fghftj • mvgmgt Central Dogma of Molecular Biology • • • • • • DNA holds the code DNA makes RNA RNA makes Protein DNA to DNA is called REPLICATION DNA to RNA is called TRANSCRIPTION RNA to Protein is called TRANSLATION Central Dogma of Molecular Biology Summary of protein synthesis • Proteins: • Chains of Amino Acids • Three nucleotide base pairs code for one amino acid. • Proteins are formed from RNA • The nucleotide code must be translated into an amino acid code. Occurs in the cytoplasm or on Rough ER RNA • Formed from 4 nucleotides, 5 carbon sugar, phosphate. • Uracil is used in RNA. – It replaces Thymine • The 5 carbon sugar has an extra oxygen. • RNA is single stranded. Translation • Translation requires: – Amino acids – Transfer RNA: (tRNA) Appropriate to its time, transfers AAs to ribosomes. The AA’s join in cytoplasm to form proteins. 20 types. Loop structure – Ribosomal RNA: (rRNA) Joins with proteins made in cytoplasm to form the subunits of ribosomes. Linear molecule. – Messenger RNA: (mRNA) Carries genetic material from DNA to ribosomes in cytoplasm. Linear molecule. Translation • Initiation— – mRNA binds to smaller of ribosome subunits, then, small subunit binds to big subunit. – AUG start codon--complex assembles • Elongation— – add AAs one at a time to form chain. – Incoming tRNA receives AA’s from outgoing tRNA. Ribosome moves to allow this to continue • Termintion— Stop codon--complex falls apart Translation • Translation requires: – Amino acids – Transfer RNA: (tRNA) Appropriate to its time, transfers AAs to ribosomes. The AA’s join in cytoplasm to form proteins. 20 types. Loop structure – Ribosomal RNA: (rRNA) Joins with proteins made in cytoplasm to form the subunits of ribosomes. Linear molecule. – Messenger RNA: (mRNA) Carries genetic material from DNA to ribosomes in cytoplasm. Linear molecule. Gene expression in bacteria • Escherichia coli (E. coli); is a Gram negative rodshaped bacterium that is commonly found in the lower intestine of warmblooded organisms. • Part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2, and by preventing the establishment of pathogenic bacteria within the intestine The LAC operon • Jacob and Monod • First scientists to elucidate a transcriptionally regulated system. They worked on the lactose metabolism system in E. Coli. • When the bacterium is in an environment that contains lactose it should turn on the enzymes that are required for lactose degradation. The LAC operon • A bacterium's prime source of food is glucose, since it does not have to be modified to enter the respiratory pathway. • So if both glucose and lactose are around, the bacterium wants to turn off lactose metabolism in favor of glucose metabolism. • There are sites upstream of the Lac genes that respond to glucose concentration. The LAC operon • beta-galactosidase: • This enzyme hydrolyzes the bond between the two sugars, glucose and galactose. • It is coded for by the gene LacZ. • Lactose Permease: • This enzyme spans the cell membrane and brings lactose into the cell from the outside environment. The membrane is otherwise essentially impermeable to lactose. It is coded for by the gene LacY. • Thiogalactoside transacetylase: • The function of this enzyme is not known. It is coded for by the gene LacA. The LAC operon • Operator (LacO) – binding site for repressor • Promoter (LacP) – binding site for RNA polymerase • Repressor (LacI) – gene encoding lac repressor protein – Binds to DNA at operator and blocks binding of RNA polymerase at promoter • Pi – promoter for LacI • CAP – binding site for cAMP/CAP complex The LAC operon • LacZ, Y, and A appear adjacent to each other on the E. Coli genome. • Preceded by a region which is responsible for the regulation of the lactose metabolic genes. • It would seem that the cell would want to turn these genes on when there is lactose around and off when lactose is absent. • The story is more complicated than that! The LAC operon • REMEMBER!!!! • A bacterium's prime source of food is glucose, since it does not have to be modified to enter the respiratory pathway. • So if both glucose and lactose are around, the bacterium wants to turn off lactose metabolism in favor of glucose metabolism. • There are sites upstream of the Lac genes that respond to glucose concentration. The LAC operon • When lactose is present, it acts as an inducer of the operon. • Enters the cell and binds to the Lac repressor, inducing a conformational change that allows the repressor to fall off the DNA. • Now the RNA polymerase is free to move along the DNA and RNA can be made from the three genes. • Lactose can now be metabolized. The LAC operon • When the inducer (lactose) is removed: • Repressor returns to its original conformation and binds to the DNA, so that RNA polymerase can no longer get past the promoter. No RNA and no protein is made. • RNA polymerase can still bind to the promoter though it is unable to move past it. • When the cell is ready to use the operon, RNA polymerase is already there and waiting to begin transcription; the promoter doesn't have to wait for the enzyme to bind. • We could say that the operon is primed for transcription upon the addition of lactose. The LAC operon • When levels of glucose (a catabolite) in the cell are high, a cyclic AMP is inhibited from forming. • glucose levels drop, more cAMP forms. • cAMP binds to a protein called CAP (catabolite activator protein), which is then activated to bind to the CAP binding site. • This activates transcription, perhaps by increasing the affinity of the site for RNA polymerase. • This phenomenon is called catabolite repression, a misnomer since it involves activation, but understandable since it seemed that the presence of glucose repressed all the other sugar metabolism operons. Genetic engineering Genetic engineering • The direct alteration of a genotype – Human genes can be inserted into human cells for therapeutic purposes – Genes can be moved from one species to another • Moving genes from human to human or between species requires the use of special enzymes known as restriction enzymes. – These cut DNA at very specific sites – They restrict DNA from another species – isolated from bacteria. Genetic engineering • Transferred DNA is denatured to give ssDNA • The probe will bind to gene of interest by Complementary base-pairing - A with T and G with C Genetically modified crops • Agrobacterium method – Uses the natural infection mechanism of a plant pathogen – Agrobacterium tumefaciens naturally infects the wound sites in dicotyledonous plant causing the formation of the crown gall tumors. – Capable to transfer a particular DNA segment (T-DNA) of the tumor-inducing (Ti) plasmid into the nucleus of infected cells where it is integrated fully into the host genome and transcribed, causing the crown gall disease. • So the pathogen inserts the new DNA with great success!!! Genetically modified crops • The vir region on the plasmid inserts DNA between the T-region into plant nuclear genome • Insert gene of interest and marker in the T-region by restriction enzymes – the pathogen will then “infect” the plant material • Works fantastically well with all dicot plant species – tomatoes, potatoes, cucumbers, etc – Does not work as well with monocot plant species - corn • As Agrobacterium tumefaciens do not naturally infect monocots Genetically modified Figure 11.21 crops •So to modify a plant: •Need to know the DNA sequence of the gene of interest •Need to put an easily identifiable maker gene near or next to the gene of interest •Have to insert both of these into the plant nuclear genome •Good screen process to find successful insertion •Clone the genetically altered plant Genetically modified crops • Can alter nutritional content – Potatoes with 21-22% more starch • Resistance to pathogens – Less damage to crops – better total yield – lower retail cost • Herbicide-resistant plants – Spraying the fields only kills weeds • Longer shelf-lives – More attractive to buy in bulk Genetically modified crops • Issues: • Destroying ecosystems – tomatoes are now growing in the artic tundra with fish antifreeze in them! • Destroying ecosystems – will the toxin now being produced by pest-resistance stains kill “friendly” insects such as butterflies. • Altering nature – should we be swapping genes between species? Genetically modified crops • Issues: • Vegetarians – what about those tomatoes? • Religious dietary laws – anything from a pig? • Cross-pollination – producing a super-weed • Human health – what of the antibiotic marker gene?