Bài giảng Biochemistry 2/e - Chapter 12: Structure of Nucleic Acids

Chapter 12Structure of Nucleic Acidsto accompanyBiochemistry, 2/ebyReginald Garrett and Charles GrishamAll 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 Outline12.1 Primary Structure of Nucleic Acids12.2 ABZs of DNA Secondary Structure12.3 Denaturation and Renaturation of DNA12.4 Tertiary Structure of DNA12.5 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 ReplicationDNA 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' directionChain Termination MethodBased 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 ddTTPChain Termination MethodMost 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 MethodRun 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 readChemical 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 residuesChemical 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 DNASecondary 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-DNAThe “canonical” base pairsSee 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-bondsMajor and minor groovesSee 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 DNASee 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-DNADiscovered 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 stacking12.4 Supercoils and CruciformsIn 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 cruciformsChromosome StructureHuman DNA’s total length is ~2 meters!This must be packaged into a nucleus that is about 5 micrometers in diameterThis 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 filamentsNucleosome StructureChromatin, the nucleoprotein complex, consists of histones and nonhistone chromosomal proteinsHistone octamer structure has been solved (without DNA by Moudrianakis, and with DNA by Richmond)Nonhistone proteins are regulators of gene expressionChemical Synthesis of Nucleic AcidsLaboratory synthesis of nucleic acids requires complex strategiesFunctional groups on the monomeric units are reactive and must be blockedCorrect phosphodiester linkages must be madeRecovery at each step must high!Solid Phase Oligonucleotide SynthesisDimethoxytrityl 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’-OHStep 2: In coupling reaction, second base is added as a nucleoside phosphoramidateSolid Phase SynthesisStep 3: capping with acetic anhydride blocks unreacted 5’-OHs of N-1 from further reactionStep 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 group12.7 Sec/Tert Structure of RNATransfer 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 tRNARibosomal RNARibosomes 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 RNASecondary structure features seem to be conserved, whereas sequence is not There must be common designs and functions that must be conserved