Sinh học - Chapter 19 (part 2): Nucleic acids

Chapter 19 (part 2)Nucleic AcidsDNA1o Structure - Linear array of nucleotides2o Structure – double helix3o Structure - Super-coiling, stem-loop formation4o Structure – Packaging into chromatinDetermination of the DNA 1o Structure (DNA Sequencing)Can determine the sequence of DNA base pairs in any DNA moleculeChain-termination method developed by SangerInvolves in vitro replication of target DNATechnology led to the sequencing of the human genomeDNA 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 4 separate rxnsEach reaction mixture contains dATP, dGTP, dCTP and dTTPEach reaction also contains a small amount of one dideoxynucleotide (ddATP, ddGTP, ddCTP and ddTTP).Each of the 4 dideoxynucleotides are labeled with a different fluorescent dye.Dideoxynucleotides missing 3’-OH group. Once incorporated into the DNA chain, chain elongation stops) Chain 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' DNA Secondary structureDNA is double stranded with antiparallel strandsRight hand double helixThree different helical forms (A, B and Z DNA.Comparison of A, B, Z DNAA: 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 A-DNAB-DNAZ-DNAZ-DNAFound 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 DNA sequence Determines Melting PointDouble Strand DNA can be denatured by heat (get strand separation)Can determine degree of denturation by measuring absorbance at 260 nm.Conjugated double bonds in bases absorb light at 260 nm.Base stacking causes less absorbance.Increased single strandedness causes increase in absorbanceDNA sequence Determines Melting PointMelting temperature related to G:C and A:T content.3 H-bonds of G:C pair require higher temperatures to denture than 2 H-bonds of A:T pair.DNA 3o StructureSuper coiling Cruciform structuresSupercoilsIn duplex DNA, ten bp per turn of helix (relaxed form)DNA helix can be over-wound.Over winding of DNA helix can be compensated by supercoiling.Supercoiling prevalent in circular DNA molecules and within local regions of long linear DNA strandsEnzymes called topoisomerases or gyrases can introduce or remove supercoilsIn vivo most DNA is negatively supercoiled.Therefore, it is easy to unwind short regions of the molecule to allow access for enzymes Each super coil compensates for one + or – turn of the double helixCruciforms occur in palindromic regions of DNA Can form intrachain base pairingNegative supercoiling may promote cruciformsDNA and NanotechnologyDNA and NanotechnologyDNA 4o StructureIn chromosomes, DNA is tightly associated with proteins Chromosome 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 proteins% major histone proteins: H1, H2A, H2B, H3 and H4Histone octamers are major part of the “protein spools” Nonhistone proteins are regulators of gene expression4 major histone (H2A, H2B, H3, H4) proteins for octomer200 base pair long DNA strand winds around the octomer146 base pair DNA “spacer separates individual nucleosomesH1 protein involved in higher-order chromatin structure.W/O H1, Chromatin looks like beads on stringSolenoid Structure of ChromatinRNASingle stranded moleculeChemically less stable than DNApresence of 2’-OH makes RNA more susceptible to hydrolytic attack (especially form bases)Prone to degradation by Ribonucleases (Rnases)Has secondary structure. Can form intrachain base pairing (i.e.cruciform structures).Multiple functionsType of RNARibosomal RNA (rRNA) – integral part of ribosomes (very abundant)Transfer RNA (tRNA) – carries activated amino acids to ribosomes.Messenger RNA (mRNA) – endcodes sequences of amino acids in proteins.Catalytic RNA (Ribozymes) – catalzye cleavage of specific RNA species.RNA can have extensive 2o structure