Determine the primary structure of nucleic acids. Structure of nucleic acids. The nucleotide sequence of DNA

Transcription

1 Determine the primary structure of nucleic acids Structure of nucleic acids 1975, determining the primary structure of nucleic acids (the nucleotide sequence) was more formidable problem than amino acid sequencing of protein (nucleic acid contain only 4 units; protein have 20) Two important breakthroughs reversed this situation --- restriction endonuclease --- polyacrylamide gel electrophoresis ( 聚丙烯醯胺膠電泳 ) The nucleotide sequence of DNA DNA is a double helical molecule Depended on the resolving power of polyacrylamide gel electrophoresis --- the chain termination or dideoxy method of F. Sanger that relies on enzymeatic replication of the DNA to be sequenced --- a base-specific chemical cleavage method developed by A. M. Maxam and W. Gilbert that exploits chemical methods for cleaving the sugarphosphate backbone of DNA strand at the location of particular bases The chain termination protocol DNA polymerase ( 聚合酶 ) dntp Template ( 模板 ) --- single strand DNA (ssdna) Primer ( 引子 ) OH 1

2 Reading dideoxy sequencing gels Secondary structures Double-stranded DNA molecules assume one of three secondary structures A, B, and Z B-DNA DNA sequencing can be fully automated 10 bp Watson-Crick base pair The DNA double helix is a stable structure H bonds Electrostatic interaction Van der waals and hydrophobic interactions Double helix structures can adopt a number of stable conformations 2

3 A-form DNA is an alternative form of right-handed DNA A-DNA Different from B-DNA --- pitch 2.46 nm bp Z-DNA is a conformational variation in the form of a left-handed double helix 3

4 The double helix is a very dynamic structure DNA behaves as a dynamic, flexible molecule Intercalating agents distort the double helix --- ethidium bromide (EtBr; 溴化乙烯 ), acridine orange (AO;Y 啶橙 ), and actinomycin D ( 釋放線菌素 ) (can insert between the stacked base pairs of DNA) Dynamic nature of the DNA double helix in solution DNA denatured Disrupt base-pairing interactions, the strands are no longer held together --- double helix denatured --- ph (>10 or <2.3) --- Temperature (melt) --- Ionic strength ( 離子強度 ) --- Hyperchromic shift (blue shift) 短波長偏移 --- DNA solution at 260 nm increases as much as 40% as the bases unstack--- Hyperchromic effect ( 增色效應 ) Melting temperature (Tm) --- G:C > A:T (H bonds) --- dependent on the ionic strength of the solution DNA renatured To re-form the duplex structure Renaturation requires reassociation of the DNA strands into a double helix, a process termed rennealing 4

5 The rate of DNA renaturation is an index of DNA sequence complexity Nucleic acid hybridization: different DNA strands of similar sequence can form hybrid duplexes The buoyant density of DNA is an index of its G:C content Tertiary structure of DNA Many DNA molecules are circular Bacterial chromosomes are covalently closed, circular DNA duplexes, as are most plasmid DNAs Plasmids --- naturally occurring --- self-replicating --- extra-chromosomal DNA molecules Supercoils are one kind of DNA tertiary structure Double-stranded circular DNA form supercoils ( 超螺旋 ) --- underwound (negatively supercoiled) --- overwound (positively supercoiled) Linking number (L) --- if DNA duplex of 400 bp, L is 40 (assuming 10 bp per turn in B-DNA) --- L = twist (T) + writhe (W) 5

6 Topoisomerases --- enzymes capable of carrying out such reactions --- change topological state of DNA --- I and II classes --- I type cut one strand of DNA double helix, pass the other strand through, and then rejoin the cut ends --- II cut both strand, pass a region of the DNA duplex between the cut ends, and then rejoin the ends --- are important players in DNA replication DNA gyrase --- is a topoisomerase of bacterial enzymes Superhelix density (specific linking difference) Toroidal supercoiled DNA --- negatively supercoiled DNA can arrange into a toroidal state Cruciforms can contribute to DNA tertiary structure --- palindromes ( 迴文 ) --- inverted repeats ( 兩端個具有相同的顛倒重複序列 ) --- cruciform ( 十字形的 ) --- negative supercoiling causes a localized distruption of hydrogen bonding between base pairs in DNA and many promote formation of cruciform --- cruciform structures have a twofold rotational symmetry about their centers and potentially create distinctive recognition sites for specific DNA-binding proteins Structure of eukaryotic chromosomes 23 pairs of dsdna molecules in the form of chromosomes The average length of which is 3 X 10 9 bp/23 or 1.3 X 10 8 nucleotide pairs Nucleosome ( 核小體 ): 146 個鹼基對的去氧核糖核酸纏繞一個八元體 ( histone) Nuclear matrix ( 細胞核基座 ) Nucleosomes are the fundamental structural unit in chromatin The DNA in a eukaryotic cell nucleus during the interphase between cell divisions exists as a nucleoprotein complex called chromatin The protein of chromatin fall into two classes --- histones Are abundant and play an important role in chromatin structure --- nonhistone chromosomal proteins 6

7 Pairs of histones H2A, H2B, H3, and H4 aggregate to form octameric core structures, and the DNA helix is wound about these core octamers, creating nucleosomes Chemically synthesized nucleic acid DNA synthesizers or gene machines are capable of carrying out the synthesis of oligonucleotides of 150 bases or more Phosphoramidite chemistry is used to form oligonucleotides from nucleotides Gene can be chemically synthesized 7

8 Secondary and tertiary structure of RNA RNA molecules are typically single-stranded Intrastrand base pairing ( 十字形構造 ) Stem-loop structures U-turns, Tetraloops, Bulges (internal loops), Junctions Secondary structure of trna 8

9 Tertiary structure of trna Secondary of rrna Comparison of rrna from various species Tertiary structure of rrna Tertiary structure of rrna 9

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