Essential knowledge 3.A.1:

1. Essential knowledge 3.A.1: DNA, and in some cases RNA, is the primary source of heritable information.

2. iv. tRNA brings the correct amino acid to the correct place on the mRNA. • v. The amino acid is transferred to the growing peptide chain. • vi. The process continues along the mRNA until a “stop” codon is reached. • vii. The process terminates by release of the newly synthesized peptide/protein. • d. Phenotypes are determined through protein activities. • To foster student understanding of this concept, instructors can • choose an illustrative example such as: • • Enzymatic reactions • • Transport by proteins • • Synthesis • • Degradation • e.

3. Genetic information is transmitted from one generation to the next through DNA or RNA. Genetic information is stored in and passed to subsequentgenerations through DNA molecules and, in some cases, RNAmolecules.

4. RNA World Hypothesis • The RNA world hypothesis proposes that self-replicating ribonucleic acid (RNA) molecules were precursors to current life. • RNA stores genetic information like DNA, and catalyzes chemical reactions like an enzyme protein. • Many viruses also store and transmit RNA

5. Chromosomes • Noneukaryotic organisms have circular chromosomes, while eukaryotic organisms have multiple linear chromosomes, although in biology there are exceptions to this rule. linear circular

6. Prokaryotes, viruses and eukaryotes can contain plasmids, which are small extra-chromosomal, double-stranded circular DNA molecules.

7. The proof that DNA is the carrier of genetic information involved a number of important historical experiments. These include: i. Contributions of Watson, Crick, Wilkins, and Franklin on the structure of DNA ii. Avery-MacLeod-McCarty experiments iii. Hershey-Chase experiment

8. The Great Debate Which chemical is used to store and transmit genetic information? Protein or DNA Most Scientists of the day(early and mid 1900(s) agreed that the substance must be protein.

9. Evidence for DNA as genetic material • Griffith, 1928 - In his work with Streptococcus pneumoniae, Griffith realized that some “transforming” agent was exchanged between bacteria which enabled to acquire traits from one another. • The use of heat to inactivate cells suggested that the agent was not protein. • This phenomenon is now called transformation - a change in phenotype by taking genetic material from the environment.

10. Griffith Experiment

11. Avery-MacLeod-McCarty experiments • Took Griffiths experiment a step further by isolating different chemical to see which one would transform bacteria. • Avery, et al., 1944 - isolated various chemicals from bacteria and used them to try transform bacteria. Only DNA worked.

12. Viruses are made of nucleic acid and protein

13. Hershey Chase Experiment (1952)

14. Chargaff (1947) • Adenine pairs Thymine; Cytosine pairs Guanine • If a mixture made from cells contained 20% Adenine, then what is the percentage of Guanine?

15. Structure of DNA • Wilkins and Franklin used X-ray diffraction to attempt to find the structure of DNA.

16. The Structure of DNA was discovered • Watson and Crick (1953) • Double Helix • Sides: phosphate and sugar • Rungs: nitrogenous bases held together by hydrogen bonds

17. Phosphate Group O O=P-O O 5 CH2 O N Nitrogenous base (A, G, C, or T) C1 C4 Sugar (deoxyribose) C3 C2 DNA Nucleotide – Monomer of DNA and RNA

18. A or G T or C Nitrogenous Bases • Double ring PURINES Adenine (A) Guanine (G) • Single ring PYRIMIDINES Thymine (T) Cytosine (C)

19. 5 O 3 3 O P P 5 5 C O G 1 3 2 4 4 2 1 3 5 O P P T A 3 5 O O 5 P P 3 DNA Strands are Anti-parallel

20. Structure of DNA

21. DNA replication ensures continuity of hereditary information. • Replication is a semiconservative process; that is, one strand serves as the template for a new, complementary strand.

22. DNA Replication (Semiconservative Model)

23. Origin of Replication • Origin of replication (“bubbles”): beginning of replication • Replication fork: ‘Y’-shaped region where new strands of DNA are elongating • Helicase:catalyzes the untwisting of the DNA at the replication fork • DNA polymerase:catalyzes the elongation of new DNA

24. DNA Replication • Antiparallel nature: • sugar/phosphate backbone runs in opposite directions (Crick); • one strand runs 5’ to 3’, while the other runs 3’ to 5’; • DNA polymerase only adds nucleotides at the free 3’ end, forming new DNA strands in the 5’ to 3’ direction only

25. DNA Replication • Leading strand: synthesis toward the replication fork (only in a 5’ to 3’ direction from the 3’ to 5’ master strand) • Lagging strand: synthesis away from the replication fork (Okazaki fragments); joined by DNA ligase (must wait for 3’ end to open; again in a 5’ to 3’ direction) • Initiation: Primer (short RNA sequence~w/primase enzyme), begins the replication process

26. Similar to ATP!! • The energy to add new nucleotides comes from the substrates themselves which are nucleoside triphosphates. • The loss of two phosphates from the substrate provides the energy to drive the reaction.

27. Retroviruses reverse the normal flow of genetic information • Genetic information in retroviruses is a special case and has an alternate flow of information: from RNA to DNA, made possible by reverse transcriptase, an enzyme that copies the viral RNA genome into DNA. • This DNA integrates into the host genome and becomes transcribed and translated for the assembly of new viral progeny.

28. RNA

29. RNA Differs from DNA 1. RNA has a sugar ribose DNA has a sugar deoxyribose 2. RNA contains the base uracil (U) DNA has thymine (T) 3. RNA molecule is single-stranded DNA is double-stranded

30. Structure of RNA

31. Three Types of RNA . • Messenger RNA (mRNA) carries genetic information to the ribosomes • Ribosomal RNA (rRNA), along with protein, makes up the ribosomes • Transfer RNA (tRNA) transfers amino acids to the ribosomes where proteins are synthesized

32. The 4th Type of RNA! The role of RNAi includes regulation of gene expression at the level of mRNA transcription. • RNA interference (RNAi) is a biological process in which RNA molecules inhibit gen expression, typically by causing the destruction of specific mRNA molecules.

33. Protein Synthesis

34. Central Dogma • Genetic information flows from a sequence of nucleotides in a gene to a sequence of amino acids in a protein.

35. The Triplet Code • The genetic instructions for a polypeptide chain are ‘written’ in the DNA as a series of 3-nucleotide ‘words’ • Codons • ‘U’ (uracil) replaces ‘T’ in RNA

36. Name the Amino Acids • GGG? • UCA? • CAU? • GCA? • AAA?

37. Transcription • The enzyme RNA-polymerase reads the DNA molecule in the 3' to 5' direction and synthesizes complementary mRNA molecules that determine the order of amino acids in the polypeptide.

38. Transcription • RNA polymerase: pries DNA apart and hooks RNA nucleotides together from the DNA code • Promoter region on DNA: where RNA polymerase attaches and where initiation of RNA begins • Terminator region: sequence that signals the end of transcription • Transcription unit: stretch of DNA transcribed into an RNA molecule

39. Transcription • Initiation~ transcription factors mediate the binding of RNA polymerase to an initiation sequence (TATA box) • Elongation~ RNA polymerase continues unwinding DNA and adding nucleotides to the 3’ end • Termination~ RNA polymerase reaches terminator sequence

40. mRNA Modifications • In eukaryotic cells the mRNA transcript undergoes a series of enzyme-regulated modifications. • Addition of a poly-A tail • Addition of a GTP cap • Excision of introns

41. mRNA modification • 1) 5’ cap: modified guanine; protection; recognition site for ribosomes • 2) 3’ tail: poly(A) tail (adenine); protection; recognition; transport • 3) RNA splicing: exons (expressed sequences) kept,introns (intervening sequences) spliced out; spliceosome

42. Translation • Translation of the mRNA occurs in the cytoplasm on the ribosome. • In prokaryotic organisms, transcription is coupled to translation of the message. Translation involves energy and many steps, including initiation, elongation and termination.

43. Translation • mRNA from nucleus is ‘read’ along its codons by tRNA’santicodons at the ribosome • tRNAanticodon (nucleotide triplet); amino acid

44. Translation • rRNAsite of mRNA codon & tRNAanticodon coupling • P siteholds the tRNA carrying the growing polypeptide chain • A siteholds the tRNA carrying the next amino acid to be added to the chain • E site discharged tRNA’s

45. Translation • Initiation~union of mRNA, tRNA, small ribosomal subunit; followed by large subunit • Elongation~•codon recognition •peptide bond formation •translocation • Termination~‘stop’ codon reaches ‘A’ site • Polyribosomes:translation of mRNA by many ribosomes (many copies of a polypeptide very quickly)

46. Mutations: genetic changes in a cell • Point mutations…. • Changes in 1 or a few base pairs in a single gene • Base-pair substitutions: •silent mutations no effect on protein •missense∆ to a different amino acid (different protein) •nonsense ∆ to a stop codon and a nonfunctional protein • Base-pair insertions or deletions: additions or losses of nucleotide pairs in a gene; alters the ‘reading frame’ of triplets~frameshift mutation • Mutagens: physical and chemical agents that change DNA

47. Genetic engineering techniques can manipulate the heritable information of DNA and, in special cases, RNA. • • Electrophoresis • • Plasmid-based transformation • • Restriction enzyme analysis of DNA • • Polymerase Chain Reaction (PCR)

48. Restriction Enzymes Analysis • A restriction enzyme (or restriction endonuclease) is an enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites. • Fragments are separated using gel electrophoresis

49. Electrophoresis • The separation of DNA fragments according to charge and size. • Smaller fragments migrate through the jell at a faster rate than larger fragments. • The gels are stained an viewed under UV light.

50. PCR • The polymerase chain reaction (PCR) is a biochemical technology in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. • Developed in 1983 by KaryMullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications