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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Chapter 12
Structure of Nucleic Acidsto 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
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Primary StructureSequencing 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
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
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'
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Chemical Cleavage MethodNot 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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Chemical Cleavage MethodFour 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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Chemical Cleavage MethodReading 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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
The ABZs of DNA
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
The “canonical” base pairs
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Major and minor grooves
See Figures 12.10, 12.11 • 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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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!
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
12.3 Denaturation of DNASee 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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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!
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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