Biochemistry Chapter 19: Nucleic Acids

Nucleosomes

-chromatin: DNA plus proteins that package SNA in more compact form --30 nm fibers in diameter --unfolds in low ionic strength (<5mM) --beads (nucleosomes) on a strong (DNA) -histones: major proteins of chromatin --eukaryotes contain 5 histones (H1, H2A, H2B, H3, H4) --small basic proteins containing numerous lysine and arginine residues whose positive charges allow the proteins to bind to the negatively charges sugar phosphate backbone of DNA -packaging of DNA into nucleosomes reduces length of DNA molecule by 10 fold -chromatin attached to RNA protein scaffold which reduced length of DNA by additional 200-fold -bacterial DNA attached to a scaffold in large loops of about 100 kb (nucleoid)

Denaturation of DNA

-completely unwinding and separation of completely single strands -in vitro processes -heat of chaotropic agent (urea of guanidinium chloride) causes denaturation -melting point Tm: temperature at which half of the DNA becomes single stranded -hyperchromicity of DNA occurs when the DNA duplex is denatured -ultraviolet light can measure degree of separation --single stranded DNA absorbs 12-40% more light at 260 nm --melting curve: plot of A260 vs temperature -denaturation is a cooperative process --unwinding begins with a bubble of single stranded DNA --bubble expands as adjacent base pairs are destabilized -GC polymers denature at might higher temperature than AT polymers -typical DNA molecule contains double-stranded regions that are G/C-rich and local single stranded regions (bubbles) that are A/T rich

Histone composition of nucleosome

-one histone H1 -two os histones H2A, H2B, H3, and H4 (histone octamer) -200 base pairs of DNA -8 histone subunits: nucleosome core particle --2H2A and H2B dimers --2HE and H4 dimers --1 3/4 turn of DNA/core particle -histones can be acetylated: decreases positive charge (histone acetylases remove acetyl groups)

Nucleotides

-phosphorylated derivative of nucleoside -ribonucleosides has 3 hydroxyl groups --2' phosphate --3' phosphate --5' phosphate -deoxyribonucleosides contain 2 hydroxyl groups --3' phosphate --5' phosphate -monolucleotides --AMP: adenosine 5'-monophosphate --GMP: guanosine 5'-monophosphate --UMP: uridine 5'-monophosphate --CMP: cytidine 5'-monophosphate --dTMP: deoxythymidine 5'-monophosphate --anionic at physiological pH --phosphorylated to di- and triphosphates -polymers of nucleotides can be abbreviated for nucleoside with and one letter abbreviation for nucleoside

Nucleotides are joined by 3'--> 5' phosphodiester linkages

-primary structure of a nucleic acid is the sequence of its nucleotide residues -polynucleotide chains have directionality --one end is 5' (no residues attached to its 5'-carbon) --other end is 3' (no residues attached to its 3'-carbon) -structural abbreviations are assumed to read 5'-->3' -nucleic acid-polyanions at physiological pH -directionality of DNA: 5' ---> 3'

Nucleic acid bases

-pyrimidine bases --uracil (RNA) --thymine (DNA) -cytosine -purine bases --adenine --guanine -heterocyclic bases can exist in at least 2 tautomeric forms --adenine and cytosine (amino or imino) --guanine, thymine and uracil can exist in either the lactam (keto) or lactim (enol) forms --amino and lactam tautomers more stable -bases can hydrogen bond --guanine, cytosine and thymine can form 3 hydrogen bonds --adenine can form 2 hydrogen bonds

Types of RNA

-ribosomal RNA: 80% of total RNA and are integral part of ribosomes -transfer RNA: 15% of total RNA and carry activated amino acids to ribosomes to be incorporated into growing polypeptide chain -messenger RNA: 3% of total RNA and encode the sequences of amino acids in proteins (carry information and least stable) -small RNA: have catalytic function -RNA: single stranded/ secondary structure

Properties of nucleases

-some act both on RNA and DNA -ribonucleases act on ONLY RNA -deoxyribonucleases act on only DNA -classified as exonucleases or endonucleases -exonucleases hydrolyze phosphodiester linkage from only one end of a polynucleotide chain --most common 3'--> 5' --5'--> 3' exonuclease also exist -endonucleases hydrolyze phosphodiester linkages at various sites within a polynucleotide chain

DNA can be supercoiled

-supercoils restore 10.4 base pairs/turn of double helix -most circular DNA molecules in cells are supercoiled -linear DNA molecules contain locally suPercoiled regions -bacterial chromosome has about 2 supercoils/1000 base pairs of DNA -topoisomerases: enzymes that add or remove supercoils enzymatically -negative/positive supercoiling: equilibrium -most DNA in cell is negatively supercoiled = easy to unwind short regions of DNA

B-DNA model

-proposed by Watson and Crick in 1953 -based on known structures of nucleosides -based on X-ray diffraction patterns that Rosalind Franklin and Maurice Wilkins obtained from DNA fibers -model could explain DNA replication and transcription

Weak forces stabilize double helix

-stacking interactions: stacked base pairs form van der Waals contacts -hydrogen bonds: H bonding between bp -hydrophobic effects: purine and pyrimidines in the interior of helix -charge-charge interactions: electrostatic repulsion of phosphate groups lessened by magnesium ions and proteins -double stranded DNA more stable than single stranded DNA

Chemical structure of nucleic acids

-sugars --ribose --deoxyribose -bases

Conformations of double stranded DNA

-B-DNA is not the only DNA conformation -A-DNA is a dehydrated form of DNA --Rosalind Franklin (1952) -more tightly wound than B-DN --major and minor grooves = similar in width --11 base pairs/turn --base pair tilted about 20 degrees -Z-DNA is a left handed DNA --no grooves and occurs in G/C regions --deoxyguanylate residues have a different sugar conformation (3'endo) and base syn

Nucleosides

-B-N-glycosicic bond connects C-1 of the sugar to N-1 of the pyrimidine of N-9 of the purine -ribonucleosides --adenosine: ribonucleoside containing adenine --guanosine: ribonucleoside containing guanine --cytidine: ribonucleoside containing cytosine --uridine: ribonucleoside containing uracil -deoxyribonucleosides --deoxyadenosine: deoxyribonucleoside containing adenine --deoxyguanosine: deoxyribonucleoside containing guanine -deoxyuridine: deoxyribonicleoside containing uracil --deoxycytidine: deoxyribonucleoside containing cytosine --thymidine: deoxyribonucleoside containing thymine -two relatively stable conformations around the glycosidic bonds of nucleotides --syn --anti

Alkaline hydrolysis of RNA

-DNA lacks 2' hydroxyl group- no cleavage -RNA treated with 0.1 M NaOh at room temperature is degraded to a mixture of 2'- and 3' nucleoside monophsphates -intermediate in reaction of RNA with base is 2',3' cyclic nucleoside monophosphate

Two antiparallel strands form a double helix

-DNA molecules consist fo 2 strands -guanine pairs with cytosine (3 H bonds) -adenine pairs with thymine (2 H bonds) -strands run in opposite directions (antiparallel) -two strands wrap around each other to form a two-stranded helical structure (double helix) -"ladder" twisted into a helix -base pairs stacked above each other to create a hydrophobic interior -two hydrophillic sugar-phosphate backbones wind around the outside of the helix (exposed to aqueous environment) -double helix has 2 grooves --major groove --minor groove -B-DNA --right handed helix --diameter= 2.37 nm --rise of the helix= 0.33 nm --pitch (complete turn) of helix= 3.40 nm --10.4 base pairs/turn -angle of rotation between adjacent nucleotides within each strand= 34.6 degrees -length of double stranded DNA molecules expressed in terms of base pairs --E. coli chromosome= 4600 base pairs --human genome= 3.2 x 10^9 base pairs

DNA is double stranded

-Erwin Chargaff found that equimolar amounts of G an C or A and T within a cell -total mole % of G + C in species vary -total mole % of G + C may vary considerably from total mole % of A + T

Restriction endonucleases

-act on DNA -restrict expression of foreigN DNA hundreds of restriction endonucleases -methylation of adenine of cytosine residues protects host DNA -type 1: catalyze methylation of host DNA and cleavage of unmethylated DNA at a specific recognition sequence -type II: cleave double-stranded DNA at or near an unmethylated recognition sequence (no methylase activity) endonucleases -recognition sites have two fold axis of symmetry (palindrome) -EcoR1: one of the first enzymes discovered --staggered cleavage --binds tightly to DNA --binds to specific DNA sequence -use of restriction endonucleases --restriction maps --DNA fingerprints --recombinant DNA: used to cut DNA to clone genes

Ribonuclease A

-bovine pancreatic ribonuclease A --single, 124 amino acid chain --cross linked by 4 disulfide bridges --pH optimum= 6 --cleaves 5'--.3' (5' hydroxyl group and 3' nucleoside monophosphate result) -structure --3 ionic amino acid residues in active site (Lys-41, His-12, Lis-119) --uses proximity effects, acid base catalysis and transition state stabilization during catalysis --pyrimidine binding pocket