LS3-4-10 - Lecture 4 DNA - Topology & Chromatin...

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Unformatted text preview: Lecture 4 DNA - Topology & Chromatin Structure Allison: p 30-35 p 37-46 and RNA Structure Allison: p 54-61 DNA Structure in a Cell -some molecules are linear e.g. most eukaryotic chromosomes Some bacteriophage & viruses -some molecules are circular (covalently closed) e.g. most Eubacterial chromosomes Mitochondria, chloroplasts Episomes (plasmids) some bacteriophage & viruses 3’ 5’ 3’ 5’ Chromatin structures in Eubacteria Bacterial chromosome ~1 mm long - Folded into Nucleoid Plasmid DNA 1 μm Fig 3.6 & 3.7 Agarose gel electrophoresis of plasmid DNA Most DNA in the cell is supercoiled Topology of covalently closed circular DNA Relaxed Supercoiled Topoisomerase – winds and relaxes DNA Increasing Topoisomerase Increasing supercoiling DNA in the cell is negatively supercoiled Topology of covalently closed circular DNA Relaxed Supercoiled Twist = number of helical turns @ 10(.5) bp per turn Writhe = number of times that double helix crosses over itself Twist + Writhe = Linking number Topology of underwound circular DNA 360 bp Twist = # helical turns (10 bp per turn) =36 Writhe = 0 Linking number (Twist +Writhe) = 36 =36 = -4 = 32 =32 =0 = 32 Topoisomerase I creates single-strand break Changes linking number by 1 Topoisomerase I acts through a covalent intermediate Topoisomerase II creates double-strand break Changes linking number by 2 Requires ATP DNA is coated by protein in the cell -protein can induce supercoiling DNA condensed minichromosome extended minichromosome Fig 3.8 Simian Virus 40 (SV40) Chromosome Structure of Mitotic Chromosomes Giemsa Stain Light Micrograph Scanning Electron Micrograph Chromatid = 700 nm diameter Fig 3.2 Long linear DNA is tightly wound Nucleosomes induce negative supercoil Micrococcal nuclease cleavage of Chromosomes ~450 bp ~300 bp ~150 bp Fig 3.3 Each histone has a core region that folds into a “histone fold” and a flexible N-terminal tail H2A and H2B form a dimer; H3 and H4 form a tetramer see Fig 3.5 The nucleosome is assembled by binding DNA to H3-H4tetramer --followed by binding two H2A-H2B dimers see Fig 3.5 Nucleosomes Consist of 8 core Histone proteins Cover 146 bp of DNA + 1 Linker Histone (H1) Covers ~180-bp DNA 10 nm Fiber Fig 3.2 Structure of Chromosomes 10nm Fibers coil into 30 nm Fibers 30 nm Fibers Fig 3.2 Structure of Chromosomes 30 nm Fibers fold into 200 nm DNA loop domains 700 nm chromatid 200 nm Loops 30 nm Fibers Fig 3.2 Central Dogma of Molecular Biology Transcription Translation DNA RNA Protein Different types of RNA in the cell - Informational mRNA messenger RNA—used as template for protein translation, considered as a linear structure tRNA transfer RNA—reads the genetic code, i.e. brings amino acids into translation reaction. tRNA has a unique clover-leaf structure Different types of RNA in the cell - Structural & Catalytic rRNA ribosomal RNA— Ribosomes are the protein synthesis factory. rRNA is large and has complex secondary/ tertiary structure. Is associated with many proteins. 7SL RNA Used in protein translocation (signal recognition particle) Different types of RNA in the cell - Catalytic snRNA small nuclear RNA-used for mRNA splicing snoRNA small nucleolar RNA used for modifying RNA (methylation, pseudouridylation) RNase P Used for tRNA Processing Telomerase Used for replicating telomeres Three major RNA types in Eubacterial gene expression Fig 4.14 Five major RNA types during Eukaryotic gene expression Fig 4.14 Different types of RNA in the cell - Regulatory “Antisense” RNA complementary to mRNA miRNAs microRNAs siRNAs small interfering RNAs RNA contains ribose (2’-OH)… RNA DNA The 2’-OH group makes RNA unstable in alkali. Alkali can attack the phosphodiester bonds and break the RNA chain. The 2’-OH can be methylated. … and Uracil instead of Thymine RNA is often single stranded RNA is a single strand polynucleotide composed of 4 ribonucleotides: A, C, G, and U. Fig 4.1 U can base-pair with A to form 2 H bonds just like A base-pairs with T in DNA Fig 4.3 G:U base pair can also form in RNA GU wobble; Fig 4.3 Higher order forms of RNA Secondary Structure Tertiary Structure Fig 4.5 RNA chains fold back on themselves to form a double helix Hairpin loop - minimum of 4 nucleotides Stem Like A-form DNA - 2’-OH prevents formation of B-form - Major groove is deep and narrow Fig 4.2 RNA can tolerate non-complementary nucleotides Bulges in stem Loops One side only Both sides of stem - opposite each other RNA secondary structure Fig 4.2 Pseudoknot (kissing loop) Base pairing is not confined locally - can be to distant parts of molecule Disease Box 6.3 Dyskeratosis congenita - defect in Telomerase pseudoknot Fig 4.7 ...
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