The 4 S s Size Solubility Shape Stability

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Rosamond Woods
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1 Lecture 24 (11/13/17) Reading: Ch1; Ch8; Ch9; , 346 Problems: Ch8 (text); 6,7,8,10 Ch8 (study-guide: applying); 1,3 Ch8 (study-guide: facts); 10,11 Ch9 (text); 1,2,3,4 Ch9 (study-guide: facts); 1,2,3,4,5 Ch24 (study-guide: facts); 3,5,6 Ch26 (text); 3 Ch26 (study-guide: applying); 2,3 Ch26 (study-guide: facts); 7 Ch27 (text); 1,2,3,4 EXT Reading: Ch9; Ch25; , Problems: Ch9 (study-guide: applying); 1,2 Ch9 (study-guide: facts); 7,8 Ch25 (text); 1-3,5-7,9,10,13-15 Ch25 (study-guide: applying); 1,4 Ch25 (study-guide: facts); 3,4,6 ucleic cids. The 4 S s 1. Size 2. Solubility 3. Shape a. - b. Z- c. Topology i. Packaging ii. Supercoiling iii. Topoisomerases 4. Stability a. ucleotides i. Tautomers ii. cid/base b. ucleic cids i. Chemistry ii. enaturation iii. Stability iv. ucleases B. Structure of the Information 1. Exceptions to flow 2. Structure 3. Levels of Control C. Recombinant : Biochemical Basis of Biotechnology 1. Restriction enzymes, ligase 2. Vectors and Inserts to make recombinant (r) 3. Transformation of hosts 4. Selection of transformants 5. Expression 6. Site-directed mutagenesis The 4 S s Size Solubility Shape Stability 1

2 Tautomeric Forms of Bases Uracil (keto) Cytidine (amine) Uracil (enol) Cytidine (imine) If during replication, an is in the imino tautomer, if will be complementary to the pyrimidine C rather than T... R T R R = CCEPTR = R G C R GGGTTTTTTGGG CCCTCCC Tautomeric Forms of Bases * 5 - GGGTTTTTTGGG CCCTCCC -5 GGGTTTGTTTGGG CCCCCCC Mutagenesis Replication Imino tautomer of GGGTTTTTTGGG CCCCCCC Replication GGGTTTTTTGGG CCCTCCC 2

3 Tautomeric Forms of Bases Modified bases can stabilize the rare tautomer enol tautomer of U (enol) R T R G C R R cid/base Properties of Bases t p 7, all bases are neutral. They can fit into middle of B- without repulsions But, acidic or basic treatment will protonate or deprotonate these bases. t low p, 7 of guanine, 1 of adenine, and 3 of cytosine become protonated. t high p, 1 of Guanine and 3 of Uracil (Thymine) become deprotonated. 3

4 cid/base Properties of Bases Charged bases can de-stabilize the glycosidic bond and mutagenesis pk a estabilizes glycosidic bond, leads to P-site * pk a Leads to à G mutations * P-site refers to a-purinic or a-pyridinic site ther Chemical Changes of Bases amage: oxidation alkylation degradation 4

5 ther Chemical Changes of Bases ther Chemical Changes of Bases eamination of C yields U, which leads to C à T mutations 5

6 ther Chemical Changes of Bases Frequency ther Chemical Changes of Bases Point-mutation by xidation: itrous cid eamination of C yields U, which leads to C à T mutations eamination of yields yp, which leads to à G mutations 6

7 ther Chemical Changes of Bases 8-xoguanine favors the syn conformation, which leads to 8 -G: bp (G à T mutations) ther Chemical Changes of Bases and other bases 7

ther Chemical Changes of Bases Methylation Some methylations are damaging. Methylation of G at 6 can basepair with C or T, which causes G à mutations Some methylations are important.

Methyl group lie in the major groove and can be used in the interaction with interaction proteins.

8 ther Chemical Changes of Bases Methylation Some methylations are damaging. Methylation of G at 6 can basepair with C or T, which causes G à mutations Some methylations are important. Methylation for endonuclease restriction recognition or protect from digestion by endonuclease. Methyl group lie in the major groove and can be used in the interaction with interaction proteins. Importance of methylation in replication: it is used to differentiate between the new and old strand. If there is a mutation, the repairing system will use the methylated strand as the template. In mammalian system, the promoter region has regular CpG content. Methylation at these sites can switch off eukaryotic gene expression. ther Chemical Changes of Bases lkylating gents 8

s the nucleoside inosine, it can base-pair with both

9 ther Chemical Changes of Bases Problem ypoxanthine is an oxidized (deaminated) derivative of adenine. s the nucleoside inosine, it can base-pair with both cytidine and adenosine. raw the structures of these base-pairs. I 9

10 I I eamination of yields yp, which leads to à G mutations C I eamination of yields yp, which leads to à T mutations I ormally, this / pattern is in the pyrimidine pattern (T), or the first part of the G pattern (//). 10

11 Stability of the Polymers: ucleic cids cid/base treatment of In Base: deprotonation at G (1) & U (3) destabilizes the glycosidic bond, which leads to P-sites. In cid: protonates at (1), C (3), & G (7). For C & G, this also destabilizes the glycosidic bond, which leads to Psites P-sites can lead to cleavage of the phosphodiester bond cid/base treatment of R Similar generation of P sites, except importantly in Base: complete cleavage of the phosphodiester bonds! Stability of the Polymers: ucleic cids Base treatment of R Base Base Base 2 + Base 11

12 Stability of the Polymers: ucleic cids Base treatment of R Base Base Base 2 + Base Stability of the Polymers: ucleic cids enaturation ds ss cid/base puts charges on bases, which causes internal repulsions and breaks up the double helix This denaturation can also be accomplished using heat. 12

This makes a nice observable ( 260 ) and perturbable (heat). What will such a plot of 260 vs. Temp. look like?

13 Stability of the Polymers: ucleic cids enaturation UV Spectrum: ative vs. enatured 40% increase This denaturation causes a hyperchromic shift. This makes a nice observable ( 260 ) and perturbable (heat). What will such a plot of 260 vs. Temp. look like? Stability of the Polymers: ucleic cids enaturation Melting Curve Like protein denaturation, this is a cooperative process. This T m value is dependent on a number of parameters. The shape is dependent on the sequence and size. 13

14 Stability of the Polymers: ucleic cids enaturation Stability of the Polymers: ucleic cids enaturation This T m value is dependent on: [salt] solvents (urea, formamide, guanidine salts) G:C content of sequence 1x SSC (Salt-Sodium Citrate) 14

Stability of the Polymers: ucleic cids Renaturation ss ds lso called re-annealing or hybridization epends on conditions (temp, [salt], solvent) that are maintained BELW the T m value.

15 Stability of the Polymers: ucleic cids Renaturation ss ds lso called re-annealing or hybridization epends on conditions (temp, [salt], solvent) that are maintained BELW the T m value. In addition, the proper formation of the complete, pristine, double helix (completely double-stranded) requires the proper amount of TIME and CCETRTI of nucleic acid. Plots of this are called C o t curves, which are much like T m curves. C o t values are dependent on the complexity of the sequence. ot enough time, you get scrambled structures Given enough time, very specific annealing occurs. Partially Renatured Stability of the Polymers: ucleic cids Renaturation EXMPLE: Karyotyping Fluorescence in situ hybridization (FIS) FIS using fluorescence probe for gene on chromosome 21. This FIS probe (a fluorescent ) found its complementary sequence among 3,000,000,000 bp in each cell! 15

16 Stability of the Polymers: ucleic cids Renaturation EXMPLE: Karyotyping Fluorescence in situ hybridization (FIS) FIS using fluorescence probe for gene on chromosome 21. This FIS probe (a fluorescent ) found its complementary sequence among 3,000,000,000 bp in each cell! Stability of the Polymers: What is responsible for STBILITY of ds? C:C nearly as stable as :U oogstein bp is very stable G:G more stable than :U and as much as G:C 16

17 Stability of the Polymers: What is responsible for STBILITY of ds What forces operate? If it s not the -bonds, then what is it? Ionic/electrostatics (salt-bridges)? It s a poly-anion; so charges actually de-stabilize ydrophobic? Unlike proteins, where this is the driving force, experiments show that the ss ds reaction is enthalpy driven process; bonds van der Waals? Yes! This is the most important. s uniform bp come together due to complementarity, the planer bases stack on each other and are close enough (<2 Å) to generate induced dipoles. nce started, it zips together as long as there are complementary bp being formed. Stacking energy = stability -bonds in complementary bp = specificity Stability of the Polymers: What is responsible for STBILITY of ds So, if its stacking energy, why are G:C rich sequences more stable than :T rich sequences? Stacking Energies in B- denine ring stacking 17

18 Stability of the Polymers: Biochemical Enzymes that catalyze the hydrolysis of the phosphodiester bonds: These enzymes are called ucleases Like proteases, if they cleave in the middle, they are called endonucleases (e.g., restriction endonucleases) If they cleave at the ends, they are called exonucleases Exonucleases can be specific for either 5 -ends or 3 -ends, or either double-stranded or single-stranded nucleic acids (e.g., S1 nuclease) lso specificity for either (ases) or R (Rses) Rases are very stable, ases require Mg +2 cofactor Can be inhibited by EPC or ET, respectively 18

Chapters 7 & 19 - Nucleosides, Nucleotides and Nucleic Acids

Chapters 7 & 19 - Nucleosides, Nucleotides and Nucleic Acids hapters 7 & 19 - ucleosides, ucleotides and ucleic Acids ucleosides, nucleotides and nucleic acids function as vitamins and coenzymes, energy carriers, second messengers, catalysts, and as genetic information