Decoding DNA: Journey from Nucleic Acids to Proteins

1. Lecture 5: Nucleic Acids into Protein. (Ch 12 and 13) Goals • Introduction to nucleic acids, DNA and replication • Understand how to make a protein (transcription) Key Terms: DNA, RNA, nucleic acid, replication, polymerase, ligase, transcription, translation, ribosome, splicing, mRNA, tRNA,

2. Introduction to Nucleic Acids

3. Mystery of the Hereditary Material • Originally believed to be an unknown class of proteins • Thinking was • Heritable traits are diverse • Molecules encoding traits must be diverse • Proteins are made of 20 amino acids and are structurally diverse

4. Structure of the Hereditary Material • Experiments in the 1950s showed that DNA is the hereditary material • Scientists raced to determine the structure of DNA • 1953 - Watson and Crick proposed that DNA is a double helix

5. Hershey-Chase Experiment • 1917, Discovery of Bacteriophage • Why doesn’t everyone die of bacterial dysentary? • What’s eating the bacteria? • The MSU story • 1950, Harold Sadoff U of Ill: Micro aerosols • 1952, Hershey and Chase Experiment Bacteriophage

6. Bacteriophages • Viruses that infect bacteria • Consist of protein and DNA • Inject their hereditary material into bacteria bacterial cell wall plasma membrane cytoplasm

7. Hershey & Chase’s Experiments • Created labeled bacteriophages • Radioactive sulfur • Radioactive phosphorus • Allowed labeled viruses to infect bacteria • Asked: Where are the radioactive labels after infection?

8. virus particle labeled with 35S virus particle labeled with 32P Hershey and Chase Results • Label protein or DNA with radio isotopes • Infect bacteria with phage particles • Sheer off the phage (blender) • Separate bacteria and phage protein • Progeny of the phage bacterial cell (cutaway view) label outside cell label inside cell

9. Hershey and Chase Results • Conclusions: • DNA is the infective material not protein • Strong inference: DNA is genetic information

10. Radical New View of Life! Sickle Cell Anemia, A Molecular Disease, Pauling et al. Science 1949

11. Structure of Nucleotides in DNA • Each nucleotide consists of • Deoxyribose (5-carbon sugar) • Phosphate group • A nitrogen-containing base • Four bases • Adenine, Guanine, Thymine, Cytosine

12. Nucleotide Bases ADENINE (A) GUANINE (G) phosphate group deoxyribose THYMINE (T) CYTOSINE (C)

13. Composition of DNA • Chargaff showed: • Amount of adenine relative to guanine differs among species • Amount of adenine always equals amount of thymine and amount of guanine always equals amount of cytosine A=T and G=C

14. Rosalind Franklin’s Work • Was an expert in x-ray crystallography • Used this technique to examine DNA fibers • Concluded that DNA was some sort of helix

15. DNA is a very long molecule and does not naturally form long, thin fibres. But it is possible to extract DNA from cells in the form of a viscous gel; if a needle is dipped into the gel and slowly wound up, it drags out a DNA fibre in which many molecules are lined up parallel to each other. The X-ray patterns given by DNA fibres show a pair of strong arcs along their vertical axis; Astbury realised that their position indicated a very regular periodicity of 3.4 along the axis of the fibre and that this figure was similar to the thickness of the DNA bases; he therefore suggested that the bases were stacked on top of each other "like a pile of pennies". He was quite right, but the well-known double helix structure had to await much better X-ray pictures (obtained by Wilkins, Franklin and colleagues at King's College, London) and the realisation by Crick and Watson (in Cambridge) that the bases were in pairs, joining two backbones running in opposite directions.

16. Watson-Crick Model • DNA consists of two nucleotide strands • Strands run in opposite directions • Strands are held together by hydrogen bonds between bases • A binds with T and C with G • Molecule is a double helix

17. Watson-Crick Model Covalent Bonds Hydrogen Bonds

18. DNA Structure Helps Explain How it Duplicates • DNA is two nucleotide strands held together by hydrogen bonds • Hydrogen bonds between two strands are easily broken • Each single strand then serves as template for new strand

19. DNA Replication • Each parent strand remains intact • Every DNA molecule is half “old” and half “new” new old old new

20. Base Pairing During Replication Each old strand serves as the template for complementary new strand

21. Enzymes in Replication • Enzymes unwind the two strands • DNA polymerase attaches complementary nucleotides • DNA ligase fills in gaps • Enzymes wind two strands together

22. A Closer Look at Strand Assembly Energy for strand assembly is provided by removal of two phosphate groups from free nucleotides newly forming DNA strand one parent DNA strand

23. Continuous and Discontinuous Assembly Strands canonlybe assembled in the 5’ to 3’ direction

24. DNA Repair • Mistakes can occur during replication • DNA polymerase can read correct sequence from complementary strand and, together with DNA ligase, can repair mistakes in incorrect strand • DNA damage from environmental factors

25. Cloning • Making a genetically identical copy of an individual • Is cloning new? • Natural Clones- Maternal twins • Synthetic Clones- • Researchers have been creating clones for decades • Clones were created by embryo splitting VIDEO CLIP

26. Dolly: Cloned from an Adult Cell • Showed that differentiated cells could be used to create clones • Sheep udder cell was combined with enucleated egg cell • Dolly is genetically identical to the sheep that donated the udder cell

27. More Clones • Mice • Cows • Pigs • Goats • Guar (endangered species)

28. Bacteriophages • Viruses that infect bacteria • Consist of protein and DNA • Inject their hereditary material into bacteria bacterial cell wall plasma membrane cytoplasm

29. Nucleic Acids Into Proteins

30. Steps from DNA to Proteins Same two steps produce ALL proteins: 1) DNA is transcribed to form RNA • Occurs in the nucleus • RNA moves into cytoplasm 2) RNA is translated to form polypeptide chains, which fold to form proteins

31. Three Classes of RNAs • Messenger RNA (mRNA) • Carries protein-building instruction • Ribosomal RNA (rRNA) • Major component of ribosome • Transfer RNA (tRNA) • Delivers amino acids to ribosome

32. DNA Bases RNA Bases Thymine Base (T) Uricil Base (U) DNA vs. RNA

33. Base Pairing During Transcription • A new RNA strand can be put together on a DNA region according to base-pairing rules • As in DNA, C pairs with G • Uracil (U) pairs with adenine (A)

34. Transcription & DNA Replication • Like DNA replication • Nucleotides added in 5’ to 3’ direction • Unlike DNA replication • Only small stretch is template • RNA polymerase catalyzes nucleotide addition • Product is a single strand of RNA

35. Promoter • A base sequence in the DNA that signals the start of a gene • For transcription to occur, RNA polymerase must first bind to a promoter

36. Gene Transcription DNA to be transcribed unwinds transcribed DNA winds up again mRNA transcript RNA polymerase

37. Adding Nucleotides 5’ 3’ growing RNA transcript 5’ 3’ direction of transcription

38. snipped out snipped out Transcript Modification unit of transcription in a DNA strand 3’ 5’ exon intron exon intron exon transcription into pre-mRNA poly-A tail cap 5’ 3’ 5’ 3’ mature mRNA transcript

39. Genetic Code • Set of 64 base triplets • Codons • Nucleotide bases read in blocks of three • 61 specify amino acids • 3 stop translation

40. Code Is Redundant • Twenty kinds of amino acids are specified by 61 codons • Most amino acids can be specified by more than one codon • Six codons specify leucine • UUA, UUG, CUU, CUC, CUA, CUG

41. Redundant?(Genetic Code Secret Decoder Ring)

42. Three Stages of Translation Initiation Elongation Termination

43. Key Players in Translation • Ribosome- Center of action • The tRNAs • Start Codon (Met) • The tRNAs- big cast • The mRNA- translated script • Stop codon

44. Ribosomes tunnel small ribosomal subunit large ribosomal subunit intact ribosome

45. Binding Sites on Large Subunit binding site for mRNA A (second binding site for tRNA) P (first binding site for tRNA)

46. tRNA Structure codon in mRNA anticodon in tRNA tRNA molecule’s attachment site for amino acid amino acid OH

47. Initiation • Initiator tRNA binds to small ribosomal subunit • Small subunit/tRNA complex attaches to mRNA and moves along it to an AUG “start” codon • Large ribosomal subunit joins complex

48. Elongation • mRNA passes through ribosomal subunits • tRNAs deliver amino acids to the ribosomal binding site in the order specified by the mRNA • Peptide bonds form between the amino acids and the polypeptide chain grows

49. Elongation

50. Termination • A stop codon in the mRNA moves onto the ribosomal binding site • No tRNA has a corresponding anticodon • Proteins called release factors bind to the ribosome • mRNA and polypeptide are released