CH 10: Molecular Biology of the Gene DNA  RNA  Protein

Sections Covered with my titles for each section 10.1: DNA as the genetic material 10.2/3: Structure of DNA and RNA 10.4/5: DNA replication : Transcription and translation 10.15: review 10.16: Mutations

History of DNA  DNA as the genetic material Griffith (1928) Griffith (1928) Found that the genetic component of pathogenic bacterial cells was not destroyed when the cells were heatedFound that the genetic component of pathogenic bacterial cells was not destroyed when the cells were heated He did not follow-up on what that component was and how/why it survived.He did not follow-up on what that component was and how/why it survived. Griffith ExperimentGriffith ExperimentGriffith

DNA as the Genetic Material  Avery (1944) Most believed protein to be genetic material at this time. Most believed protein to be genetic material at this time. Avery found that pathogenic bacterial cells treated with protein digesting enzymes could still transform harmless bacterial cells. Avery found that pathogenic bacterial cells treated with protein digesting enzymes could still transform harmless bacterial cells. Cells treated with a DNA digesting enzyme could not. Cells treated with a DNA digesting enzyme could not.

DNA as the Genetic Material  Avery (1944) Avery concluded that DNA and not protein must be the genetic material. Avery concluded that DNA and not protein must be the genetic material. Many refused to accept this conclusion. Many refused to accept this conclusion. Thought his findings only applied to bacteria and not eukaryotic cells.Thought his findings only applied to bacteria and not eukaryotic cells.

DNA as the Genetic Material  Hershey-Chase Experiment (~1950) Their work confirmed to the scientific community that DNA was the genetic material. Their work confirmed to the scientific community that DNA was the genetic material. Considered an “elegant” experiment. Considered an “elegant” experiment. Very simple and demonstrates a great deal.Very simple and demonstrates a great deal. See page 183 See page 183

Hershey-Chase Experiment  They took advantage in a chemical difference between DNA and protein DNA contains the elements: C, H, O, N, P DNA contains the elements: C, H, O, N, P Protein contains the elements: C, H, O, N, S Protein contains the elements: C, H, O, N, S

Hershey-Chase Experiment  Experiment utilized bacteriophages Bacteriophages are viruses that infect bacteria. Bacteriophages are viruses that infect bacteria. Knew that a virus’ genetic material enters the host cell Knew that a virus’ genetic material enters the host cell as a result the bacterial cell makes more virus as directed by the virus’ genetic materialas a result the bacterial cell makes more virus as directed by the virus’ genetic material

Hershey-Chase Experiment  More on viruses….. Viruses have two components: Viruses have two components: An outer protein coat with nucleic acid insideAn outer protein coat with nucleic acid inside

Hershey-Chase Experiment The Experiment 1. Allowed one sample of viruses to infect bacteria grown on a radioactive (RA) sulfur-35 medium Viruses made had RA Sulfur-35 in their protein coats. Viruses made had RA Sulfur-35 in their protein coats.

Hershey-Chase Experiment The Experiment 2. Allowed another sample of viruses to infect bacteria grown on a radioactive (RA) phosphorus-32 medium Viruses made had RA phosphorus-32 in their DNA. Viruses made had RA phosphorus-32 in their DNA.

Hershey-Chase Experiment 3. The two RA viral cultures were isolated and each was allowed to infect a new (non RA) bacterial culture. Exp’t was done in a liquid medium called the supernatant. Exp’t was done in a liquid medium called the supernatant. 4. Cultures were gently shaken in a blender to shake the virus off of the outside of the bacteria.

Virus infecting bacterial cell

Hershey-Chase Experiment 5. Each culture was centrifuged to separate the liquid medium (supernatant) from the infected bacteria. 6. The bacteria and the supernatant were checked for radioactivity. Whatever entered the bacteria is the genetic material. Whatever entered the bacteria is the genetic material.

Hershey-Chase Experiment What they found: Bacteria infected with the virus with a RA S- 35 (protein) coat Bacteria infected with the virus with a RA S- 35 (protein) coat The infected bacteria were NOT RAThe infected bacteria were NOT RA The supernatant was RAThe supernatant was RA This is evidence that the protein did not enter the bacteria and thus, could not be the genetic material. This is evidence that the protein did not enter the bacteria and thus, could not be the genetic material.

Hershey-Chase Experiment For the bacteria infected by virus with RA P- 32 in their DNA For the bacteria infected by virus with RA P- 32 in their DNA The infected bacteria were RAThe infected bacteria were RA The supernatant was not RAThe supernatant was not RA This is evidence that the DNA entered the bacteria and thus, MUST be the genetic material.This is evidence that the DNA entered the bacteria and thus, MUST be the genetic material. ey.phphttp:// ey.php

Structure of DNA What was known about DNA Chemical components are: Chemical components are: Deoxyribose – 5 carbon sugarDeoxyribose – 5 carbon sugar Phosphate groupsPhosphate groups Nitrogenous basesNitrogenous bases Adenine Adenine Guanine Guanine Cytosine Cytosine Thymine Thymine

Structure of DNA  Nitrogenous bases were of 2 types: Purines: have a double-ring structure Purines: have a double-ring structure Adenine (A)Adenine (A) Guanine (G)Guanine (G) Pyrimidines: have a single-ring structure Pyrimidines: have a single-ring structure Cytosine (C)Cytosine (C) Thymine (T)Thymine (T) Page 185Page 185

Structure of DNA  Chargaff’s findings (1949) Studied DNA from many organisms Studied DNA from many organisms Found that the amount of guanine is always equal to the amount cytosine and the amount of adenine is equal to the amount of thymine. Found that the amount of guanine is always equal to the amount cytosine and the amount of adenine is equal to the amount of thymine. G=CG=C A=TA=T

Structure of DNA  X-Ray Crystallography Data on DNA Maurice Wilkins and Rosalind Franklin Maurice Wilkins and Rosalind Franklin Franklin’s data suggested that DNA was a long thin molecule of 2 nm diameterFranklin’s data suggested that DNA was a long thin molecule of 2 nm diameter Data also indicated a repeating pattern consistent with a helix.Data also indicated a repeating pattern consistent with a helix. Wilkins shared Franklin’s data and lab notes with Watson and Crick without her permission.Wilkins shared Franklin’s data and lab notes with Watson and Crick without her permission.

Rosalind Franklin  As a scientist Miss Franklin was distinguished by extreme clarity and perfection in everything she undertook. Her photographs are among the most beautiful X-ray photographs of any substance ever taken. Their excellence was the fruit of extreme care in preparation and mounting of the specimens as well as in the taking of the photographs. -- J. D. Bernal [1958 N] [1958 N] [1958 N]

Franklin’s X-Ray Data

Structure of DNA "The instant I saw the picture my mouth fell open and my pulse began to race.... the black cross of reflections which dominated the picture could arise only from a helical structure... mere inspection of the X-ray picture gave several of the vital helical parameters." Watson

Structure of DNA  In 1953 Watson, Crick, and Wilkins put the pieces together and proposed their famous double helix structure for DNA. Watson, Crick, and Wilkins were awarded a Nobel Prize for deciphering the structure of DNA Watson, Crick, and Wilkins were awarded a Nobel Prize for deciphering the structure of DNAWilkins

Watson and Crick

Structure of DNA  DNA is a double-stranded helix Each strand is a long chain of covalently bonded nucleotides Each strand is a long chain of covalently bonded nucleotides Phosphates can bond to carbon 5 or carbon 3 of deoxyribosePhosphates can bond to carbon 5 or carbon 3 of deoxyribose Phoshpates link the sugars to form the backbone of the chain Phoshpates link the sugars to form the backbone of the chain Bases bond to carbon 1 of deoxyriboseBases bond to carbon 1 of deoxyribose Page 187Page 187

Structure of DNA  Each strand has a 5’ and a 3’ end Two DNA strands run in opposite directions Two DNA strands run in opposite directions One runs 5’  3’ and the other 3’  5’One runs 5’  3’ and the other 3’  5’

Structure of DNA  The two strands are joined by hydrogen bonds between the bases Two H bonds form between A and T.Two H bonds form between A and T. Three H bonds form between G and C.Three H bonds form between G and C.

G C A T

Structure DNA

DNA Replication  DNA replication – DNA synthesis Occurs in the nucleus during ___ of the cell cycle Occurs in the nucleus during ___ of the cell cycle Goal is to make an exact copy of the cell’s DNA Goal is to make an exact copy of the cell’s DNA Put another way -- goal is to duplicate the chromosomes.Put another way -- goal is to duplicate the chromosomes. Replication

Semi-Conservative Model  Each newly made piece of DNA is ½ old DNA and ½ new DNA (page 188) Simple Simple animation of replication

DNA Replication-enzymes needed Helicases Helicases Open the H bonds between the strandsOpen the H bonds between the strands Stabilizing proteins Stabilizing proteins Hold the two strands apartHold the two strands apart

DNA Replication: enzymes needed DNA polymerase III DNA polymerase III Adds nucleotides to the 3’ end of DNAAdds nucleotides to the 3’ end of DNA Say…synthesizes DNA in the 5’  3’ directionSay…synthesizes DNA in the 5’  3’ direction It cannot initiate (start) a new DNA strandIt cannot initiate (start) a new DNA strand DNA polymerase I DNA polymerase I Removes primer sequences and fills in the gaps with DNARemoves primer sequences and fills in the gaps with DNA Other DNA polymerases Other DNA polymerases Proofread the DNA and correct mutationsProofread the DNA and correct mutations

DNA Replication-enzymes needed “Primer” enzyme – not shown in text “Primer” enzyme – not shown in text Starts synthesis in the 5’  3’ directionStarts synthesis in the 5’  3’ direction Makes a primer sequence to which DNA polymerase III can add DNAMakes a primer sequence to which DNA polymerase III can add DNA DNA ligase DNA ligase Joins newly made DNA segments after the primer sequences have been removed and replaced by DNA polymerase I.Joins newly made DNA segments after the primer sequences have been removed and replaced by DNA polymerase I.

DNA Replication 1. Helicases and stabilizing proteins open and unwind small sections of DNA and hold the strands apart. Occurs at specific locations on the DNA – called origins of replication Occurs at specific locations on the DNA – called origins of replication 2. Primer enzymes synthesize primer strands in the 5’  3’ direction on each DNA strand.

DNA Replication 3. DNA polymerase III adds DNA to each primer sequence in the 5’  3’ direction.

DNA Replication 4. Proteins open more of the DNA (replication fork opens more). 5. DNA synthesis continues in the 5’  3’ direction on one strand. (leading strand) Another primer is laid down on the other strand and then DNA synthesis continues. (lagging strand) Another primer is laid down on the other strand and then DNA synthesis continues. (lagging strand)

Primer sequences

DNA Replication 6. Process continues until all of the DNA has been replicated. 7. Primer sequences are cut out, the gaps filled in with DNA 8. DNA ligase joins the new DNA sequences.