2 Did you know?100 years ago we did not know why some children had brown eyes and some blue75 years ago we did know the structure of dextrose50 years ago we did not know the correct number of chromosomes25 years ago we did not know any of the genes linked to cancerWhat is one you know of?BRCA breast cancer gene
3 Differences Between Eukaryote and Prokaryote Cell EukaryotesNucleusMembrane-bound organellesMicrotubules are the building blocks for a flagellaCell membranes can contain cholesterolCell size is usually biggerIntrons in the DNAProkaryotesNo nucleusNo membrane-bound organellesFlagella is the building blockNo cholesterol in cell membranesCell size is usually smallerUsually no introns
5 Sources of DNA How are Eukaryotes and Prokaryotes different? DNA is in the nucleusProkaryotes have no nucleus, so where is the DNA?Floating in the cytoplasm, which is usually attached to the cell membrane.Bacteria contain 1 long circular DNA molecule, super coiled.E. coli contains 1 chromosome w/4000 genes and 4.6 million base pairs (bp)R Plasmids: bacteria with small ring of DNA floating in cytoplasm and these contain the antibiotic resistance genesGenes are turned “on” or “off” easily
6 Eukaryotic DNA DNA packaged into chromosomes Each single DNA may contain several million nucleotides and many thousands of genesHumans have 46 chromosomes per cell with about 3 billion base pairs making up about 40,000 genes
7 Historical Figures in Molecular Biology (visit DNAi.org) MiescherGriffithAvery, McCarty and MacLeodChargaffWilkins, Franklin, Watson & CrickMendel in 1866 determined inheritance in pairs1867 Miescher called DNA nuclein, acidic substance in cell nuclei1928 Griffith1944 Avery, McCarty, and MacLeod determine DNA molecule responsible for heredity1950 Chargaff determined the ration of AT and CG are equal although the quantity of DNA varies between species1953 Wilkins, Franklin, Watson and Crick determine 3D structure of DNA
8 Key people in genetics and DNA Gregor Mendel: heredity passed down from parents; relationship between phenotype and genotypeSchleiden and Schwann’s “cell theory” explained fertilization of sperm and egg to make zygote1905 discovered sex chromosomes existed; years later realized there are more chromosomes that are responsible for traitsWatson and Crick: 3D structure of DNA in 1953DNA actually discovered in 1869, by 1900 understood that it was composed of 5C sugar, phosphate, and 5 types of nitrogen rich bases (ATCGU), 1920s we understood that RNA and DNA were different
9 Key Historical People Erwin Chargaff Rosalind Franklin determined percents of purines and pyrimidines presentRosalind FranklinX-ray diffraction technique was key to understanding the helix structure
10 Role of X-Ray Crystallography X-rays diffracted by the regularly arranged atoms of a simple crystal (Max von Laue)Pauling, Franklin, Wilkins, Watson and Crick were all working diligently to discover the structure of DNA
11 What Nucleotides are involved? What are the 4 nucleotides of DNA?Adenine, thymine, guanine, cytosineWhat pairs with what?Adenine and ThymineCytosine and Guanine
12 The three parts of the nucleotide building block of DNA are the sugar, the base and the phosphate. The complex of the sugar with the base is called a nucleoside.SugarThe sugar is the 5-carbon sugar deoxyribose. By convention the carbons on this sugar are labeled 1' through 5'.PhosphateThe phosphate is attached to the 5' carbon of the deoxyribose sugar.BaseThe base is attached to the 1' carbon of the deoxyribose sugar. There are four different bases found in DNA. Because each base contains at least two nitrogen atoms, they are called nitrogenous bases. There are two classes of bases, the pyrimidines (cytosine (C) and thymine (T)), and the purines (adenine (A) and guanine (G)).
13 Complementary Base Pairing DNA consists of two polynucleotide chains wound around each other to form a double helix. The two chains are held together by complementary base pairing; that is, specific bonding between A and T bases and between G and C bases on the two strandsTwo antiparallel DNA polynucleotide chains held together by complementary base pairing. To make a stable double helix, the two strands of DNA are antiparallel; that is, the 5’ - 3' direction of one strand runs opposite to the other strand. The two DNA chains are held together by complementary base pairing between A and T bases and between G and C bases.The helix has a right hand twist.
14 In a DNA polynucleotide chain, nucleotides are joined by phosphodiester bonds formed between the 5' carbon of one sugar and the 3' carbon of the next sugar. A free phosphate defines the 5' end of the chain and a free hydroxyl group defines the 3' end of the chain.
15 1. 3’ end 2. phosphate 3. 5’ end 4. thymine 5. 3’ end 6 1. 3’ end 2. phosphate 3. 5’ end 4. thymine 5. 3’ end 6. phosphodiester bond7. cytosine 8. deoxyribose 9. guanine 10. 5’ end11. adenine
16 Basics to DNA Structure Rails of ladder: run in opposite directions (anti-parallel)Contains alternating units of deoxyribose sugar and phosphateEach rung composed of a base pair held together by weak hydrogen bonds10 base pairs per turn34 A total so 3.4 A between pairs
18 DNA Replication DNA helicase Single-strand binding proteins Primase DNA polymeraseDNA ligaseOkazaki fragments
19 So why is DNA replication so important to us? DNA is the carrier of genetic information for all living organismsThrough the process of replication, the entire genome is copied and passed down to each new cell made in the body.Replication is also the way genetic information is passed from parents to offspring.
20 ReplicationDNA polymerase can only directly synthesize new DNA in the 5' to 3' directionChargaff’s Rule for determining how many nucleotides are present:In double-stranded DNA, G = C, and A = T.If C = 21, then G = 21 and G + C is 42. Therefore A + T = = 58, and T = 58/2 = 29 percent.A/T, G/C, and (A+G)/(C+T) are all equal to 1Semiconservative Replication: making 2 daughter stands from a single parent strandTherefore DNA replication takes place prior to cell division
21 DNA Helicase and SSB DNA Helicase is an enzyme which begins the unzipping process. Also prevents DNAfrom rebinding.Problem is that it creates a knotted up mess of DNATopoisomerase cuts one strand of unwound and allows it to unwind and then reseals it. It prevents damage to the DNA by allowing it to swivel.Once DNA is unzipped the base pairs of each single stand will begin forming helix structureSSB (single strand binding proteins) are formed to block this action; prevent recombining
23 Replication BubbleThe DNA begins to split from many points along the strands and separate from that point, creating a bubble-like area.
24 Replication ForkWhen referring to the replication of DNA in a singular direction, the original DNA splits in two, forming two prongs, which resemble a fork.
25 Primase and DNA Polymerase Primase is an RNA polymerase which does not need a primer to initiate synthesisRNase H comes into remove the RNA primer made by primase before DNA is replicatedDNA polymerase III can only add nucleotides onto the 3’ end of an existing DNA fragment so if this is the case then where does the first piece of DNA come from?
26 Okazaki fragments and DNA Ligase DNA synthesis is always 5’ to 3’Leading strand is synthesized and the lagging strand has small fragments formed which are later joined together.Fragments are called Okazaki fragments after the scientist who discovered this process.Polymerase I removes RNA primer and replaces it with DNA nucleotides in Okazaki fragmentsDNA ligase is the enzyme which joins the Okazaki fragments together
27 DNA ReplicationFollow the steps in order to review the process
28 Checking your knowledge What are the two strands of DNA called after they unzip?Leading and laggingWhat enzyme is used for unzipping?DNA helicaseWhat direction does DNA replicate?Actually both, 5’ to 3’ easy, continuous and self correcting; 3’to 5’ takes longer, more chance of error and requires DNA polymerase and DNA ligase
29 Checking your knowledge What are the fragments formed during replication called and what strand are they formed on?Okazawki fragments; lagging 3’ to 5’ strandWhat 3 enzymes are required for DNA replication?DNA helicase, DNA polymerase, topoisomerase
30 RNA: how is it different from DNA? Pentose sugar is ribose instead of deoxyriboseUracil replaces ThymineRNA is single stranded
31 What is transcription? Where does it occur? Transcription: the process of deciphering a DNA nucleotide code and converting into into an RNA nucleotide code; RNA carries genetic message to a ribosome for translation into a protein.Proteins do the work of cells and give cells and organisms their unique characteristics.
32 Transcription occurs in 5’ to 3’ direction Figure 2.11Note: single strand of RNA
33 Transcription Initiation Transcription factors bind to the TATA box which guide the RNA Polymerase to the starting point of the gene.
34 Transcription Elongation RNA Polymerase continues to assemble RNA nucleotides in a complementary fashion to the 3’5’ template strand
35 Transcription Termination Once the RNA Polymerase hits the termination sequence, it releases from the template and the RNAS transcript floats away.
36 RNA ProcessingSlicesome cuts out the intron sequences and joins the exons to make the final mRNAPoly A tails are added to the 3’ endMethylated G cap added to the 5’ end
37 More essential termsIntron: region on a gene that is transcribed into a mRNA molecule but not expressed in a protein; spacer DNAExon: region of a gene that directly codes for a protein, it is the region of the gene that is expressed
39 Some essential termsOperon: section of prokaryotic DNA consisting of one or more genes and their controlling elements.Promoter: the region at the beginning of a gene where RNA polymerase binds; the promoter promotes the recruitment of RNA polymerase and other factors required for transformationOperator: region on an operon that can either turn on or off expression of a set of genes depending on the binding of a regulatory moleculeGenetic engineers use promoter and operator regions to turn on / off the production of certain genes
41 What is translation and where does it occur? Translation: the process of reading the mRNA nucleotide code and converting it into a sequence of amino acids.Translation occurs in the ribosomes which are in the cytoplasmHow does this process differ in prokaryotes vs eukaryotes?
44 Translation Initiation Initiator tRNA binds to the AUG codon of the mRNA. This tRNA has an anticodon of UAC and carries Met amino acid so all translated products start with the Met amino acid
45 Translation Elongation A site – tRNA enters with its new amino acidP site – growing amino acid chain is linked to newly arriving amino acid by a peptide bondE site – tRNA leaves without its amino acid
46 Translation Termination When stop codon appears in A site, there is no tRNA to bind so a release factor binds insteadThis causes the polypeptide chain to releaseFinally, it also causes the ribosome subunits to disassociate and translation is terminated