Molecular Biology of the Gene

1. Molecular Biology of the Gene • The structure of the genetic material • DNA replication • The flow of genetic information from DNA to RNA to protein • Microbial genetics

2. DNA is the Genetic Material • In 1928, Frederick Griffith found that a killed pathogenic strain of bacteria can convert a live harmless strain • Mixed killed pathogenic bacteria with live harmless bacteria: got live pathogenic bacteria • What is the transforming factor? • Enter Hershey and Chase

3. Hershey and Chase Experiments • Used bacteriophages (viruses) to infect bacteria • Consist of viral DNA (or RNA) and protein coat- nothing more • Is it the protein or the DNA that transforms the bacteria?

5. Hershey and Chase, cont. • Viruses grown on heavy media • Heavy sulfer: tags protein • Heavy phosphorus: tags DNA • Infected bacteria with phages • Knocked off excess phages • Centrifuged the solution- cells heavy, phages light

6. Results

8. DNA and RNA • Nucleic acids: chains of nucleotide monomers • Each strand of DNA or RNA has a sugar-phosphate backbone to which a nitrogenous base is attached • Purines: • Adenine and guanine • Double ring structures • Pyrimidines: • Thymine and cytosine • Single ring structures

10. DNA and RNA • RNA very similar to DNA • Similarities: • Sugar-phosphate backbone and nitrogenous bases • Differences: • RNA usually single stranded • RNA has no thymine (uracil instead) • Ribose instead of deoxyribose

11. DNA • Structure determined by James Watson (American) and Francis Crick (British) • They used results from Maurice Wilkins and Rosalind Franklin; also Erwin Chargaff • From x-ray crystallography, Franklin knew DNA was a helix with uniform diameter (2 nm) • Sugar-phosphates were the backbone, and the nitrogenous bases were ‘inside’

12. DNA • Erwin Chargaff had determined [A] = [T] and [G] = [C] • Watson and Crick put it all together: sugar-phosphate backbone on a double-stranded helix with A-T and G-C steps • W and C published their work in 1953 in Nature • Watson, Crick, and Wilkins won Nobel Prize in 1962 (Franklin had died of ovarian cancer)

16. DNA Replication • Relationship between structure and function evident in helix • Structure dictates how it will be copied • Watson and Crick model: helix unzips, each strand has complementary bases added • Semi-conservative model result: each helix has one new strand, one old strand

19. DNA Replication • Begins at origins of replication (multiple specific sites) • Strands are anti-parallel • DNA polymerase adds only to 3’ end (so replication moves 5’ 3’) • About a dozen enzymes and proteins involved in replication • DNA polymerase, DNA ligase, etc.

23. DNA Information • DNA  RNA: transcription • RNA  Protein: translation • Beadle and Tatum: determined that mold nutritional mutants lacked specific enzymes and genes • Suggested the one gene- one enzyme idea

24. Genetic Information • DNA  RNA: transcription • Language the same: nucleic acids • Only minor differences (single stranded, ribose, uracil) • RNA  Protein: translation • Different languages (nucleic acid, protein) • 3 RNA bases in a row constitutes a codon • 1 codon codes for 1 amino acid (ie 3 RNA bases codes for 1 amino acid)

26. Codons • Translation: changing from nucleic acid language to amino acid language • 4 RNA bases have to code for 20 amino acids • Redundancy in the system (several codons code for same aa); no ambiguity (no codon codes for multiple aa’s)

29. Transcription • Occurs in nucleus • DNA unzips • 1 strand serves as a template • RNA polymerase: adds appropriate complementary bases • Promoter: • Binding site for RNA polymerase • Located at start of gene to be transcribed

31. Transcription produces genetic messages in the form of RNA • Stages: • Initiation: binding of RNA polymerase and start of synthesis • Elongation: RNA synthesis continues, RNA strands begins to peel away from DNA template • Termination: RNA polymerase gets to stop codon, and transcription ends • Makes mRNA, tRNA, rRNA

33. RNA Processing • mRNA is used in translation • Before leaving nucleus, eukaryotic mRNA is processed • 5’ cap, poly A tail • Removal of introns

34. tRNA • Single strand of RNA about 80 nucleotides in length • Several double stranded regions • One loop is called the anticodon • At the other end, aa attachment site

36. Ribosomes build polypeptides • Ribosome: • Two subunits (one large, one small) • Each subunit made up of proteins and rRNA • Prokaryotic and eukaryotic ribosomes are similar, but different • Eukaryotic ribosomes are more complex and are larger

39. Translation • Initiation: • mRNA binds to small ribosomal subunit • tRNA (methionine) binds to the mRNA • Large ribosomal subunit binds to small unit, making ribosome functional • tRNA fits into binding site on ribosome (P-site)

40. Translation • Elongation: • Anticodon of incoming tRNA molecule pairs with mRNA codon at the A-site • Polypeptide transferred from P-site tRNA to AA on A-site tRNA • tRNA in P-site leaves ribosome; tRNA in A-site moves to P-site • New tRNA enters A-site • Continues until stop codon

42. Translation • Termination: • Stop codons do not code for AAs • Polypeptide released • Ribosomal subunits separate

43. Flow of Genetic Information • Replication: DNADNA; nucleus • Transcription: DNARNA; nucleus • Translation: RNAProtein; cytoplasm • Translation is rapid: less than 1 minute in most cases • 1 mRNA is usually translated by several ribosomes at once

45. Mutations • Mutation: change in the DNA sequence • Two general types: • Base substitutions: change out one base for another • Base insertsions/deletions: add or remove bases (usually really bad) • Reading frame shifts: adding or deleting bases NOT in multiples of 3 shifts the reading frame for the rest of the protein

48. Viral DNA • Viruses: genes in a box- they are not alive • Consist of a capsid (protein coat) and genetic material (DNA or RNA) • Viruses are parasites that can only reproduce in cells • Two possible life cycles: • Lytic cycle • Lysogenic cycle

49. Lytic Cycle

50. Lysogenic Cycle