Lecture 15: 05/24/16 DN: Molecular basis of Inheritance 1
DN Double Helix 2
3
DN: Molecular basis of Inheritance Historical Overview! First Isolation of DN DN as genetic material ransformation change in phenotype by the acquisition of external DN DN vs Protein Chargaff s Rules : and G:C composition in cells Structure of DN Franklin s X-ray Diffraction photograph of DN Watson and Crick he Double Helix --------------------------------------------------------- DN Replication: Meselson and Stahl s Semi-conservative model. 4
DN: Molecular basis of Inheritance First Isolation of DN 1869: Miescher isolated nuclei White blood cells Nuclein HO O P O - O - large molecules, acidic, and rich in phosphorus salmon sperm Nucleic acids 1900s: scientist had determined that nucleic acids were made of: 5C Sugar Phosphate base Nucleotide denine Guanine hymine Cytosine Uracil (Only in RN) 5
DN: Molecular basis of Inheritance DN as genetic material ll cells contained DN By he 1930 s universally accepted: 1928: Griffith discovered the ransformation Phenomenon Studied the pathogenicity of Streptococcus pneumoniae Can the genetic trait of pathogenicity be transferred between bacteria? 6
DN: Molecular basis of Inheritance DN as genetic material Griffith (1928) Can the genetic trait of pathogenicity be transferred between bacteria? 2 Strains of S. pneumoniae Smooth (S) have a capsule - pathogenic Rough (R) no capsule non pathogenic Inject 7
DN: Molecular basis of Inheritance DN as genetic material very (1930s-40s) Isolated the RNSFORMION factor Extracted all molecular material Systematically remove or destroy various componenents RNase Protease DNase 8
DN: Molecular basis of Inheritance DN as genetic material Hershey-Chase (~1952) DN or Protein as the genetic material of bacteriophages? bacteriophages are viruses that infect bacteria DN encapsulated by proteins Colorized EM 9
DN: Molecular basis of Inheritance Life Cycle of bacteriophage Inject DN into bacteria Hijacks bacteria replication and translation machinery to replicate its phage DN and make head and tail proteins Lyse bacteria and release new bacteriophages Package replicated DN 10
DN: Molecular basis of Inheritance DN or Protein as the genetic material of bacteriophages to produce more phage? Infect bacteria Separate Bacteria and phage by mixing Grow phages with radioactive materials Centrifuge mixture 35 S labelled protein (Cys & Met a.a.) supernatent pellet 32 P labelled DN (phosphate) supernatent pellet 11
DN: Molecular basis of Inheritance Structure of DN Base Phosphate H 5 C 4 C 1 C 3 C 2 C1 is attached to base Purines and G (2 rings) Pyrimidines C and (1 ring) 5C sugar (deoxyribose) C2 is missing OH C3 is reactive site (3 end or 3 OH) C5 attached to phosphate: reactive (5 end or 5 phosphate) 12
DN: Molecular basis of Inheritance Structure of DN DN is polymer of nucleotides Condensation reaction: loss of water 5 H 3 H 5 3 13
DN: Molecular basis of Inheritance Structure of DN Chargaff s Rule Diversity of species 1. ll had DN 2. otal amounts differed BU Pecentage of and the same G and C 14
DN: Molecular basis of Inheritance Structure of DN Rosalind Franklin s Famous photograph of DN Diffraction (DN) (Collect and focus x-rays) Diffraction pattern is directly 15 correlated to the 3D shape of specimen
DN: Molecular basis of Inheritance Structure of DN Watson and Crick: DN Double Helix Very Stable WHY? Base pair across the helix (H-bonding) : G C Phosphate far apart Base which are hydrophobic: stack atop each other (van der waals) 16
DN: Molecular basis of Inheritance Structure of DN Double Helix Right-handed helix Uniform Diameter Helix has Uniform Diameter Strands are antiparallel 0.34 nm rise per nucleotide 3.4nm rise per turn (10 nucleotides per turn) 17
DN: Molecular basis of Inheritance,, G, C his is basis for amazing genetic diversity among species HOW? Central Dogma DN RN Protein 18
DN: Molecular basis of Inheritance How is DN replicated? 3 Hypothesis to est? 1. Conservative Hypothesis Parent DN helix remains intact, and a 2 nd new copy is made 2. Semi Conservative Hypothesis 2 strands of Parent DN helix separate, each function as a template to make COMPLEMENRY strands. 3. Dispersive Hypothesis Daughter DN helix get a mix of new DN. 19
DN Replication How is DN replicated? Conservative Semi-Conservative Dispersive 20
21 DN Replication C G C G C G C G G C G C C G C G G C G C G C G C Figure 16.9 a d Parent Strand: - and G-C base pairs Separation of parent strands Each parent strand becomes a template for complementary strands Nucleotides connected by phosphodiester bond 2 new DN molecules
Meselson and Stahl s Experiment : mechanism of DN Replication 1) Bacteria cultures in medium containing 15 N 2) Bacteria transferred to medium containing 14 N 3) DN sample centrifuged after 20 minutes (first replication) 4) DN sample centrifuged after 20 minutes (first replication) 2 Less dense More dense Figure 16.11 22
Meselson and Stahl s Experiment: mechanism of DN Replication First replication Second replication Conservative model Semiconservative model 14 N 15 N + 14 N Dispersive model 15 N 23
DN Replication Bacteria has 4.6 million bases pairs to replicate Human diploid cell has 3 billion base pairs to replicate 46 (long) DN molecules (Polymers) Has to be fast and accurate > 2000 bases per second are polymerized!! only 1 mistake per 10 billion bases added!! Many proteins involved! 24
DN Replication Replication Fork Replication Fork Origin of Replication - specialized site where replication begins Easy to unwind because high number of / base pairs Bacteria has 1 Eukaryotic cells - 100s to 1000s per chromosome Replication fork - Ends of replication bubble (origin) Helicase special protein which unwinds DN (uses P) Single stranded binding proteins (SSBP s) - Binds single stranded DN and keeps it from base pairing 25
DN Replication Initiation Lateral Expansion (Elongation) Fusion 26
DN Replication DN Polymerase III catalyses the addition of deoxyribose nucleotides to the 3 end (3 OH) dp, dp, dgp, dcp looks at opposite (template) strand and chooses correct base catalyses the phosphodiester bond energy source: energy released from PPi 2 Pi (14 kcal of energy) high fidelity (very seldom makes a mistake) : 1 in 10 billion base additions 27
DN Replication DN Polymerase III 28
DN Replication Primase synthesizes short (5-10 bases in length) RN primer supplies the 3 OH needed by DN Pol III to add bases DN Polymerase I - removes RN primer and replaces it with DN DN ligase joins (ligates) DN together to make one continuous strand (Note: Make sure you know able 16.1 (Campbell 7 th edition)!) 29
DN Replication DN replication only takes place in the 5 3 direction! Why? Polymerization only takes place at the 3 end (3 OH) 30
DN Replication : Lagging strand Synthesis 1 Primase joins RN nucleotides into a primer. 3 5 5 3 emplate strand 2 DN pol III adds DN nucleotides to the primer, forming an Okazaki fragment. 3 RN primer 5 1 3 5 4 3 fter reaching the next RN primer (not shown), DN pol III falls off. fter the second fragment is primed. DN pol III adds DN nucleotides until it reaches the first primer and falls off. 3 5 3 2 5 Okazaki fragment 1 1 3 3 5 5 5 DN pol 1 replaces the RN with DN, adding to the 3 end of fragment 2. 5 3 2 1 3 5 Figure 16.15 6 DN ligase forms a bond between the newest DN and the adjacent DN of fragment 1. 7 he lagging strand in this region is now complete. 5 3 2 Overall direction of replication 1 3 5 31
DN Replication: Overview Helicase SSBP 32
DN Replication: elomeres Dilemma: What about the ends of linear DN? 33
DN Replication: elomeres Dilemma: What about the ends of linear DN? elomerase is the answer! ~2500 base pairs at the end of DN is a repeat GGG CCC elomerase is a protein with a built-in RN template UCCCU dds back elomere ends 34
DN Replication: elomeres elomerase adds back nucleotides at the ends of DN Repeat again and again 35