SPU-22: The Unity of Science from the Big Bang to the Brontosaurus and Beyond

1. SPU-22: The Unity of Science from the Big Bang to the Brontosaurus and Beyond Lecture 21 21 April 2014 Science Center Lecture Hall A

2. Today’s Topics Meselson-Stahl experiment: How DNA replicates Nirenberg-Matthaei experiment: How DNA codes its message Central Dogma for cell producing proteins Some questions and facts related to cells

3. Much More To See After W&C One key question: How does DNA reproduce? Gunter Stent: Consider three possibilities - semi-conservative; fully conservative; and dispersive (what are these?)

4. Possible Methods Of DNA Replication

5. Determined Detectives: III Matthew Meselson and Franklin Stahl Q. How distinguish between 3 choices? A. Use heavy isotope of nitrogen, 15N, to enrich DNA in E. coli (model organism). Watch mass of resultant DNA generation by generation (~ 1 hr each) via centrifuging in cesium chloride solution (nearly same density as DNA)

6. How Prepare E. Coli? Take ordinary 14N E. Coli and let it reproduce with food laced with 15N After many generations, centrifuge mixture in gradated CsCl mixture Separate denser (15N) E. Coli

7. How Do Replication Experiment? Place 15N E. Coli in ordinary (14N) food After each generation (about 1 hr), centrifuge E. Coli with same type of CsCl mixture and examine relative amounts of various densities of E. Coli

8. Schematic: Meselson-Stahl Centrifuge

9. Meselson-Stahl Centrifuge: Partial Result

10. DNA Replication Method Distinguished by 15N

11. How Reach Conclusion? Semi conservative: Have smaller and smaller fraction of “half-and-half” E. Coli, with rest being pure 14N E. Coli Fully conservative: After each generation, would have smaller fraction with 15N E. Coli with rest being pure 14N E. Coli Dispersive: Have one “band,” closer and closer to pure 14N density

12. Matthew Meselson (1928- )

13. Frank Stahl (1929- )

14. How Does DNA Convey Its Message? What is DNA’s main message? Likely what proteins to make How is message conveyed? Likely via code What are elements (letters) of code? Likely base pairs (i.e., likely four letters only) What do words of code signify? Likely amino acids

15. First Proposed Code: George Gamow (Summer 1953) Four bases denote one amino acid Bases on corners of diamond, with two sides being complementary bases; bases on top and bottom from different “turns” of helix, could be any of four bases. Number of combinations = 20 (next slide)

16. Gamow’s Scheme For Code

17. Gamow’s Scheme For Code(Concluded) Brilliant: 20 is number of biological amino acids Scheme doesn’t work, either physically or chemically, as Watson and Crick realized upon receiving Gamow’s paper

18. George Gamow (1904-1968)

19. Gamow Got People Thinking How many bases needed for code? One – clearly doesn’t work (why?) Two – same But three (4x4x4 = 64) clearly allows more than enough words for 20 amino acids. But, if so, what base triplet denotes which amino acid? Not obvious!

20. Cracking Code Many scientists felt RNA responsible for making proteins based on DNA code Marshall Nirenberg, joined by his postdoc, Heinrich Matthaie who made arcane experiment work, succeeded in spring 1961 in completing experiment to identify first code for an amino acid

21. Cracking Code (Cont’d) Needed cell-free, stable -- not easy -- system (cytoplasm from E. Coli without DNA) to try to produce protein from amino acids after introduction of artificially created RNA into system. Used very simple RNA: bases UUU… only. Tried all 20 amino acids; only found one protein chain, made of only one amino acid: phenylalanine

22. Cracking Code (Cont’d) Nirenberg presented result at international conference in Moscow in summer 1961. About half dozen attendees. Electrified them; word spread; talk was then repeated at plenary session at end of conference; impressed everyone

23. Heinrich Matthaei (1929- ) And Marshall Nirenberg (1927-2010)

24. Cracking Code (Cont’d) First word decoded, although length of word (number of bases) unknown Clever experiments by Sydney Brenner & Crick led to conclusions: 1. Group of three bases (or, less likely, a multiple of three bases) codes one amino acid

25. Cracking Code (Cont’d) 2. Code is not of overlapping type (what does this mean?) 3. Sequence of bases read from fixed starting point…no special “commas” to show how to select correct triplets 4. Code is probably degenerate: In general, more than one base triplet codes for single amino acid

26. Cracking Code (Concluded) Lot of clever work by bunch of talented people yielded: full (redundant) code, plus triplets signifying starting and (redundant) ending of set of triplets for protein Finished in 1966; Nobel followed in 1968

27. Base Code For Amino Acids: I

28. Base Code For Amino Acids:II

29. What Triplet Indicates “Start”? Methionine. How use in protein? (See next slide.) Note: Read “Base Code For Amino Acids II” from inside out (e.g., CCU is proline)

30. Start And Stop Codons

31. How Do Cells Make Proteins? Crick named “Central Dogma” as answer to this question. We turn to its description – all told, a fascinating story, but for us “just so”

32. It’s an RNA World RNA plays critical role in life of cell. It comes in three main flavors: 1. Messenger RNA (mRNA) carries genetic information on amino acids from DNA within nucleus to region outside -- cytoplasm – where amino acid is manufactured 2. Transfer RNA (tRNA), one for each type of amino acid, binds and carries it to growing end of protein chain at ribosome 3. Ribosome RNA (rRNA) of ribosomes makes whole protein (see following cartoons) [World is here series of “just so” stories]

33. Cartoon Of Central Dogma

34. Cartoon Of Protein Synthesis

35. Cartoon Of Ribosome (rRNA)

36. Do We Really Know Details? My answer: No Are there good prospects of knowing? Yes. How? Probing with combined high spatial and temporal resolution

37. Do We Really Know Details?(Concluded) Attosecond (what’s that?) time resolution is almost here. But there’s an accompanying problem: digesting the information. Similarly with corresponding spatial resolution

38. Few Sample Questions Overall, who conducts orchestra and how? How do materials in syntheses know where and when to go? What controls rates at, and order in, which various cell components are made? How, in detail, does “supply side” work?

39. DNA Conundrum Should DNA length be correlated (positively) with organism complexity? One would think so, but no; it isn’t

40. DNA Conundrum(concluded) Take four examples, apparently reliable: Number of base pairs in whole (haploid) DNA: 1. Onion ~ 20 billion 2. Lungfish 133 billion 3. Salamander 120 billion 3. Human 3 billion

41. Onion

42. Lungfish

43. Salamander

44. Size Sorting: Biology’s Peppercorn Analogy How long is human being’s total of DNA? Length of DNA in 1 cell ≈ 0.34 x 10-9 x 6 x 109 ≈ 2 m (diploid number: M + F) Number of cells ≈ 0.1 x 0.2 x 2 m3/(10-5 x 10-5 x 10-5 m3) ≈ 4 x 1013 [vol body/vol cell] Length: DNA in body ≈ 1014 m ≤ 700 a.u.!