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

Biochemistry 2/e - Garrett & Grisham

Chapter 12

Structure of Nucleic Acidsto accompany

Biochemistry, 2/e

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

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 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

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

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

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

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'

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

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

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

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

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

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

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

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!

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

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

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

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

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!

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

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

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

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

Ribosomal RNA

• Secondary structure features seem to be conserved, whereas sequence is not

• There must be common designs and functions that must be conserved

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