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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 12 Structure of Nucleic Acids to accompany Biochemistry, 2/e.

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Presentation on theme: "Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 12 Structure of Nucleic Acids to accompany Biochemistry, 2/e."— Presentation transcript:

1 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 12 Structure of Nucleic Acids to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida

2 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Outline 12.1 Primary Structure of Nucleic Acids 12.2 ABZs of DNA Secondary Structure 12.3 Denaturation and Renaturation of DNA 12.4 Tertiary Structure of DNA 12.5 Chromosome Structure 12.6 Chemical Synthesis of Nucleic Acids 12.7 Secondary and Tertiary Structure of RNA

3 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

4 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Primary Structure Sequencing Nucleic Acids Chain termination method (dideoxy method), developed by F. Sanger Base-specific chemical cleavage, developed by Maxam and Gilbert Both use autoradiography - X-ray film develops in response to presence of radioactive isotopes in nucleic acid molecules

5 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company DNA Replication DNA is a double-helical molecule Each strand of the helix must be copied in complementary fashion by DNA polymerase Each strand is a template for copying DNA polymerase requires template and primer Primer: an oligonucleotide that pairs with the end of the template molecule to form dsDNA DNA polymerases add nucleotides in 5'-3' direction

6 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

7 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chain Termination Method Based on DNA polymerase reaction Run four separate reactions Each reaction mixture contains dATP, dGTP, dCTP and dTTP, one of which is P-32-labelled Each reaction also contains a small amount of one dideoxynucleotide: either ddATP, ddGTP, ddCTP or ddTTP

8 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

9 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chain Termination Method Most of the time, the polymerase uses normal nucleotides and DNA molecules grow normally Occasionally, the polymerase uses a dideoxynucleotide, which adds to the chain and then prevents further growth in that molecule Random insertion of dd-nucleotides leaves (optimally) at least a few chains terminated at every occurrence of a given nucleotide

10 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chain Termination Method Run each reaction mixture on electrophoresis gel Short fragments go to bottom, long fragments on top Read the "sequence" from bottom of gel to top Convert this "sequence" to the complementary sequence Now read from the other end and you have the sequence you wanted - read 5' to 3'

11 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chemical Cleavage Method Not used as frequently as Sanger's Start with ssDNA labelled with P-32 at one end Strand is cleaved by chemical reagents Assumption is that strands of all possible lengths, each cleaved at just one of the occurrences of a given base, will be produced. Fragments are electrophoresed and sequence is read

12 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chemical Cleavage Method Four reactions are used G-specific cleavage with dimethyl sulfate, followed by strand scission with piperidine G/A cleavage: depurination with mild acid, followed by piperidine C/T cleavage: ring hydrolysis by hydrazine, followed by piperidine C cleavage: same method (hydrazine and piperidine), but high salt protects T residues

13 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

14 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chemical Cleavage Method Reading the gels... It depends on which end of the ssDNA was radioactively labelled! If the 5'-end was labelled, read the sequence from bottom of gel to top (5' to 3') If the 3'-end was labelled, read the sequence from top of gel to bottom (5' to 3') Note that the nucleotide closest to the P-32 will be missed in this procedure

15 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

16 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

17 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

18 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The ABZs of DNA Secondary Structure See Figure for details of DNA secondary structure Sugar-phosphate backbone outside Bases (hydrogen-bonded) inside Right-twist closes the gaps between base pairs to 3.4 A (0.34 nm) in B-DNA

19 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

20 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

21 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The “canonical” base pairs See Figure The canonical A:T and G:C base pairs have nearly identical overall dimensions A and T share two H-bonds G and C share three H-bonds G:C-rich regions of DNA are more stable Polar atoms in the sugar-phosphate backbone also form H-bonds

22 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

23 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Major and minor grooves See Figures 12.10, The "tops" of the bases (as we draw them) line the "floor" of the major groove The major groove is large enough to accommodate an alpha helix from a protein Regulatory proteins (transcription factors) can recognize the pattern of bases and H- bonding possibilities in the major groove

24 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

25 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

26 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Comparison of A, B, Z DNA See Table 12.1 A: right-handed, short and broad, 2.3 A, 11 bp per turn B: right-handed, longer, thinner, 3.32 A, 10 bp per turn Z: left-handed, longest, thinnest, 3.8 A, 12 bp per turn See Figure 12.13

27 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

28 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

29 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Z-DNA Discovered by Alex Rich Found in G:C-rich regions of DNA G goes to syn conformation C stays anti but whole C nucleoside (base and sugar) flips 180 degrees Result is that G:C H-bonds can be preserved in the transition from B-form to Z-form!

30 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

31 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

32 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

33 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 12.3 Denaturation of DNA See Figure When DNA is heated to 80+ degrees Celsius, its UV absorbance increases by 30-40% This hyperchromic shift reflects the unwinding of the DNA double helix Stacked base pairs in native DNA absorb less light When T is lowered, the absorbance drops, reflecting the re-establishment of stacking

34 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

35 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

36 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

37 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

38 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

39 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 12.4 Supercoils and Cruciforms In duplex DNA, ten bp per turn of helix Circular DNA sometimes has more or less than 10 bp per turn - a supercoiled state Enzymes called topoisomerases or gyrases can introduce or remove supercoils Cruciforms occur in palindromic regions of DNA Negative supercoiling may promote cruciforms

40 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

41 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

42 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

43 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

44 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

45 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

46 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chromosome Structure Human DNA’s total length is ~2 meters! This must be packaged into a nucleus that is about 5 micrometers in diameter This represents a compression of more than 100,000! It is made possible by wrapping the DNA around protein spools called nucleosomes and then packing these in helical filaments

47 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Nucleosome Structure Chromatin, the nucleoprotein complex, consists of histones and nonhistone chromosomal proteins Histone octamer structure has been solved (without DNA by Moudrianakis, and with DNA by Richmond) Nonhistone proteins are regulators of gene expression

48 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

49 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

50 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

51 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chemical Synthesis of Nucleic Acids Laboratory synthesis of nucleic acids requires complex strategies Functional groups on the monomeric units are reactive and must be blocked Correct phosphodiester linkages must be made Recovery at each step must high!

52 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Solid Phase Oligonucleotide Synthesis Dimethoxytrityl group blocks the 5’-OH of the first nucleoside while it is linked to a solid support by the 3’-OH Step 1: Detritylation by trichloroacetic acid exposes the 5’-OH Step 2: In coupling reaction, second base is added as a nucleoside phosphoramidate

53 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

54 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Solid Phase Synthesis Step 3: capping with acetic anhydride blocks unreacted 5’-OHs of N-1 from further reaction Step 4: Phosphite linkage between N-1 and N-2 is reactive and is oxidized by aqueous iodine to form the desired, and more stable, phosphate group

55 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

56 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

57 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 12.7 Sec/Tert Structure of RNA Transfer RNA Extensive H-bonding creates four double helical domains, three capped by loops, one by a stem Only one tRNA structure (alone) is known Phenylalanine tRNA is "L-shaped" Many non-canonical base pairs found in tRNA

58 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

59 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

60 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

61 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

62 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

63 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Ribosomal RNA Ribosomes synthesize proteins All ribosomes contain large and small subunits rRNA molecules make up about 2/3 of ribosome High intrastrand sequence complementarity leads to (assumed) extensive base-pairing

64 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

65 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Ribosomal RNA Secondary structure features seem to be conserved, whereas sequence is not There must be common designs and functions that must be conserved

66 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company


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