DNA The Genetic Material

1. DNA The Genetic Material

2. 1947 Chargaff • DNA composition: “Chargaff’s rules” • varies from species to species • all 4 bases not in equal quantity • bases present in characteristic ratio • humans: A = 30.9% T = 29.4% G = 19.9% C = 19.8% RulesA = T C = G That’s interesting!What do you notice?

3. 1953 | 1962 Structure of DNA • Watson & Crick • developed double helix model of DNA • other leading scientists working on question: • Rosalind Franklin • Maurice Wilkins • Linus Pauling Wilkins Pauling Franklin

4. 1953 article in Nature Watson and Crick Watson Crick

5. Rosalind Franklin (1920-1958)

6. Discussion • Summarize: What do you remember about the chemical composition of DNA? Consider the following vocab words? • Nucleotide, hydrogen bond, double helix, deoxyribose, phosphate, nitrogenous base, adenine, cytosine, guanine, thymine, purine, pyramidine, phosphodiester bond

7. Double helix structure of DNA “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson & Crick

8. Base pairing in DNA Purines adenine (A) guanine (G) Pyrimidines thymine (T) cytosine (C) Pairing A : T 2 bonds C : G 3 bonds

9. Directionality of DNA You need to number the carbons! Think Five = Phosphate nucleotide PO4 N base 5 CH2 This will beIMPORTANT!! O 1 4 ribose 3 2 OH

10. Anti-parallel strands Nucleotides in DNA backbone are bonded from phosphate to sugar between 3 & 5 carbons DNA molecule has “direction” complementary strand runs in opposite direction 3’ and 5’ will determine where replication and transcription can begin and end 5 3 3 5

11. Bonding in DNA hydrogen bonds covalent phosphodiester bonds 5 3 3 5 ….strong or weak bonds? How do the bonds fit the mechanism for copying DNA?

12. DNA Organization • DNA is organized in long strands called chromosomes. • Circular in prokaryotes • Linear in eukaryotes

13. CHECKPOINT • Without notes, try to diagram or describe the structure of a strand of DNA, labeling all molecules, bonds, 3’ and 5’ ends. • If you can’t, memorizing that structure is your homework tonight!

14. DNA Replication 2007-2008

15. But how is DNA copied? • Replication of DNA • Ensures the continuity of genetic information • base pairing means each side will serve as a template for a new strand

16. Copying DNA Replication of DNA new strand is 1/2 parent template & 1/2 new DNA = semi-conservativecopy process

17. DNA Replication Large team of enzymes coordinates replication Let’s meetthe team…

18. Replication: 1st step Unwind DNA helicase enzyme unwinds part of DNA helix (hence “helicase,” AMAZING I KNOW) stabilized by single-stranded binding proteins helicase single-stranded binding proteins replication fork

19. Replication Fork • Replication begins at a point on the chromosome called the “origin.” • Helicase bonds to the origin, starts unzipping the strands, and moves progressively away, forming a “replication fork.” helicase

20. Replication: 2nd step • Build daughter DNA strand • add new complementary bases • Polymerization, an anabolic process • DNA polymerase III But… We’re missing something! What? But where’s theENERGYfor the bonding! DNA Polymerase III

21. Where does energy for bonding usually come from? Energy of Replication We comewith our ownenergy! energy YourememberATP!Are there other waysto get energyout of it? energy Are thereother energynucleotides?You bet! And weleave behind anucleotide! CTP ATP TTP GTP AMP ADP GMP TMP CMP modified nucleotide

22. Energy of Replication The nucleotides arrive as nucleosides DNA bases with P–P–P P-P-P = energy for bonding DNA bases arrive with their own energy source for bonding bonded by enzyme: DNA polymerase III ATP GTP TTP CTP

23. Adding bases can only add nucleotides to 3 end of a growing DNA strand need a “starter” nucleotide to bond to strand only grows 53 Replication 3 5 energy DNA Polymerase III DNA Polymerase III energy DNA Polymerase III energy DNA Polymerase III energy B.Y.O. ENERGY! The energy rulesthe process 3 5

24. Discussion • So we follow helicase along and replicate the strand in the 5’->3’ direction (that’s 5’->3’ of the strand being build, the template runs 3’->5’ because DNA is antiparallel)… But what is the problem that we have now created?

25. ligase 5 3 5 3 need “primer” bases to add on to energy  no energy to bond energy energy energy energy energy energy 3 5 3 5

26. Leading & Lagging strands Okazaki ligase 3 3 3 3 3 3 3 5 5 5 5 5 5 5 Limits of DNA polymerase III • can only build onto 3 end of an existing DNA strand  Okazaki fragments Lagging strand growing replication fork  Leading strand Lagging strand • Okazaki fragments • Short DNA fragments • joined by ligase • “spot welder” enzyme DNA polymerase III Leading strand • continuous synthesis

27. Replication fork / Replication bubble DNA polymerase III 3 3 3 3 3 3 3 3 3 3 3 growing replication fork growing replication fork 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 leading strand lagging strand leading strand lagging strand leading strand lagging strand

28. Starting DNA synthesis: RNA primers 3 3 3 3 3 3 DNA polymerase III 5 5 5 5 5 5 But there’s yet another problem! • can only build onto 3 end of an existing strand growing replication fork primase RNA RNA primer • built by primase, serves as starter sequence for DNA polymerase III • @ start of leading strand, and at start of each Okazaki fragment

29. Replacing RNA primers with DNA ligase 3 3 3 3 5 5 5 5 Ligase • Connects strands DNA polymerase I • removes sections of RNA primer and replaces with DNA nucleotides DNA polymerase I growing replication fork RNA But DNA polymerase I still can only build onto 3 end of an existing DNA strand. One primer can’t be acted upon…

30. Chromosome erosion 3 3 3 3 5 5 5 5 Houston, we have a problem! DNA polymerase I growing replication fork DNA polymerase III RNA Loss of bases at 5 endsin every replication • chromosomes get shorter with each replication • limit to number of cell divisions

31. Telomeres 3 3 3 3 5 5 5 5 Repeating, non-coding sequences at the end of chromosomes = protective cap to erode instead of gene sequence growing replication fork telomerase Telomerase • enzyme extends telomeres • can add DNA bases at 5 end • different level of activity in different cells • high in stem cells & cancers -- Why? TTAAGGG TTAAGGG TTAAGGG

32. Replication fork direction of replication DNA polymerase III lagging strand DNA polymerase I 3’ primase Okazaki fragments 5’ 5’ ligase SSB 3’ 5’ 3’ helicase DNA polymerase III 5’ leading strand 3’ SSB = single-stranded binding proteins

33. http://highered.mcgraw-hill.com/sites/0072943696/student_view0/chapter3/animation__dna_replication__quiz_1_.htmlhttp://highered.mcgraw-hill.com/sites/0072943696/student_view0/chapter3/animation__dna_replication__quiz_1_.html

34. Discussion • Summarize the functions of the DNA replication enzymes… • Helicase • DNA polymerase III • DNA polymerase I • Primase • Ligase • Telomerase

35. DNA polymerases DNA polymerase III 1000 bases/second! main DNA builder DNA polymerase I 20 bases/second editing, repair & primer removal DNA polymerase III enzyme

36. Editing & proofreading DNA 1000 bases/second = lots of typos! DNA polymerase I proofreads & corrects typos repairs mismatched bases removes abnormal bases repairs damage throughout life reduces error rate from 1 in 10,000 to 1 in 100 million bases

37. Fast & accurate! It takes E. coli <1 hour to copy 5 million base pairs in its single chromosome divide to form 2 identical daughter cells Human cell copies its 6 billion bases & divide into daughter cells in only few hours remarkably accurate only ~1 error per 100 million bases ~30 errors per cell cycle These errors = mutations, can change the type or amount of protein produced. More on that later…

38. From Gene to Protein How Genes Work

39. What do genes code for? • How does DNA code for cells & bodies? • how are cells and bodies made from the instructions in DNA DNA proteins cells bodies

40. The “Central Dogma” • Flow of genetic information in a cell • How do we move information from DNA to proteins? transcription translation RNA DNA protein trait DNA gets all the glory, but proteins do all the work! replication

41. Transcription fromDNA nucleic acid languagetoRNA nucleic acid language

42. RNA as opposed to DNA • ribose sugar • N-bases • uracil instead of thymine • U : A • single stranded • lots of RNAs • mRNA, tRNA, rRNA… transcription DNA RNA

43. Kinds of RNA • The sequence of RNA bases and structure of the RNA molecule determines its function • There are more than 100 kinds! Major ones: • mRNA - transcription product, carries info from DNA to ribosome • tRNA - translation intermediate, converts genetic info to protein sequence • rRNA - makes up ribosomes • RNAi - various RNA molecules interfere with transcription, helping control gene expression

44. Transcription • Making mRNA • transcribed DNA strand = template strand • untranscribed DNA strand = coding strand • same sequence as RNA • synthesis of complementary RNA strand • transcription bubble • enzyme • RNA polymerase or RNAP coding strand 3 A G C A T C G T 5 A G A A A C G T T T T C A T C G A C T DNA 3 C T G A A 5 T G G C A U C G U T C unwinding 3 G T A G C A rewinding mRNA template strand RNA polymerase 5 build RNA 53

45. How does RNAP “know” where to “read?” • Promoter region • RNAP binding site before beginning of gene • “Tells RNAP to start here” • Many promoters includeTATA box binding site • DNA sequence TATAAA • Enhancer region • binding site far upstream of gene • turns transcription on HIGH

46. Transcription Factors • Initiation complex • transcription factors bind to promoter region • suite of proteins which bind to DNA • turn on or off transcription • trigger the binding of RNA polymerase to DNA

47. http://www.youtube.com/watch?v=41_Ne5mS2ls RNA polymerase http://highered.mcgraw-hill.com/sites/0072507470/student_view0/chapter3/animation__mrna_synthesis__transcription___quiz_1_.html Matching bases of DNA & RNA A • Match RNA bases to DNA bases on one of the DNA strands C U G A G G U C U U G C A C A U A G A C U A 5' 3' G C C A T G G T A C A G C T A G T C A T C G T A C C G T

48. intron = noncoding (inbetween) sequence exon = coding (expressed) sequence Eukaryotic genes have junk! • Eukaryotic genes are not continuous • exons = the “real gene” • expressed / coding • introns = the “junk” • inbetween sequence intronscome out!

49. intron = noncoding (inbetween) sequence exon = coding (expressed) sequence mRNA splicing • Post-transcriptional processing • eukaryotic mRNA needs work after transcription • primary transcript = pre-mRNA • mRNA splicing • edit out introns • make mature mRNA transcript ~10,000 bases eukaryotic DNA pre-mRNA primary mRNA transcript ~1,000 bases mature mRNA transcript spliced mRNA

50. snRNPs snRNA intron exon exon 5' 3' spliceosome 5' 3' lariat 5' 3' exon exon mature mRNA excised intron 5' 3' RNA splicing enzymes • snRNPs • small nuclear RNA • proteins • Spliceosome • several snRNPs • recognize splice site sequence • cut & paste gene No, not smurfs! “snurps”