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img017 - Blackboard Learning System 2 H-bond...

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Unformatted text preview: 10/27/2010 Blackboard Learning System .......... 2. H-bond donors/acceptors in major/minor grooves .......... 3. implications for protein/DNA recognition ‘ ..... f) Major/minor grooves of A- and Z—DNA mmOl'gV’DOVC" Wow .......... 1. major edge recessed, m'nor edge exposed in A form 0? 1m. 130365 . .......... 2. major edge exposed, minor edge recessed in 2 form - é .......... 3. implications for protein/DNA recognition ..... g) RasMol views of A-, B~, and Z-DNA ..... h) In vivo relevance 9% .......... 1. A-DNA: existence in vivo is questionable (4N9 W .......... 2. B—DNA: predominant form in vivo .......... :3. 2- DNA. can be generated in vivo, may occur naturally ............... a. requires a specific sequence bmoh‘fig @010“ 1003an ............... b. favored by negative superhelical density (see Lecture 32) ............... c. stabilized by Z-DNA binding proteins ............... cl. B—Z junction: Ha et al., 2005, Crystal structure of a junction .................... between B~DNA and Z—DNA reveals two extruded bases, .................... Nature 437: 1183-1186. 0111900er File: Lecture 32 Lecture 32 BCMB 4010/6010 Lectures Fall, 2009 6 DNA 1) Reading Assignment ..... a) Garrett and Grisham, Chapter 11, Structure of Nucleic Acids _ 2) n tu a 'on of dsD A g ..... a) Dena uration y eat (Figures 11.18 and 11.19) .......... 1. denaturation (melting): conversion of dsDNA duplex to two ssDNA chains .......... 2. conveniently monitored by measuring absorbance at 260 nm ............... a. A260 ssDNA (unstacked bases) = 1. 4 x A260 dsDNA (stacked bases) ............... b. hyperchromic shift of approximately 40% as dsDNA is denatured .......... 3. cooperative process L WW bP unjiaflc W fibS’l“ @ZXOHM ............... a. relatively sharp transition ............... b. Tm = midpoint of transition . .......... 4. Tm is dependent on GC content and salt concentration GI ‘ C ............... a. Tm (in °C) = 0. 41 (%GC) + 16. 6 log [Na+] + 81. 5 .. . » I. -mseeverlm M. mteractsob) ............... c. salt shields charge repulsion of ionized phosphoryl groups 4 30‘ H Nice/962- ..... b) Denaturation by pH {,me PliOIS OF ”61559-3 .......... 1. protonation of bases occurs at acid pH (< 2.3) aha/19?; piiorfllnipiiilin .......... 2. deprotonation of bases occurs at alkaline pH (> 10) .......... 3. either treatment disrupts H-bonds and denatures the duplex 4“ .......... 4. alkali is preferred because acid promotes hydrolysis of the glycosidic linkage ..... c) Denaturation by solvents that stabilize the unfolded form .......... 1. urea, mm dismpt I-Ivctvorahom i‘HWawom ~ 3) Renaturation and Hybridization of DNA pwftll’g DNH back Logging ..... a) Renaturation = reannealing (Figure 11.19) .......... 1. two step process ........... .a. nucleation (second order, slow) @/ ........... .b. zippering (first order, fast) .......... 2. optimum rate occurs at approximately 20 °C below Trn ..... b) Hybridization = annealing ...uga.edu/.../cobaltMainFrame.dowebct 3/6 ...
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