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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 32887-6777 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
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
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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
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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
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
Secondary Structure See Figure 12.10 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
See Figure 12.10 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
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
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!
See Figure 12.17 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
Chromosome Structure
Human DNAs 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
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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
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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
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
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Ribosomal RNA
Secondary structure features seem to be conserved, whereas sequence is not There must be common designs and functions that must be conserved