lecture 20 - DNA/RNA structure

what is the basic buliding block of DNA and what is it made of

nucleotides and they are made of a phosphate group, pentose sugar, and base

carbon numbering in pentose sugar

why is DNA and RNA negatively charged?

The phosphate group is an acid. At physiological pH the phosphate group of every nucleotide within the DNA/RNA strand is deprotonated and thus carries a net negative charge

what are DNA and RNA polymers called

nucleic acids

• bc of the acidic phosphate groups

difference in pentose sugar in DNA vs. RNA

structure of bases

whats the bond between pentose sugar and base

what is a nucleoside

base & sugar (not phosphate)

Deoxyribonucleotides

difference between thymine and uracil

Ribonucleotides

DNA primary structure

nucleotide sequence

how is nucleotide sequence written and read?

• 5’ → 3’

• pACGTA (p = 5’ end)

what is a short nucleotide called

oligonucleotide

• only a few residues (nucleotides)

5’ and 3’ ends

• free phosphate at 5’ end

• hydroxyl at 3’ end

is DNA asymmetric or polar

yes its both

DNA secondary structure

DNA base-pairing

• base-pairing means hydrogen bonding between the bases in the two DNA strands

• pairing is specific based on H-bond complementarity: guanine forms three H-bonds with cytosine, GoC; adenine forms two H-bonds with thymine, A=T (or uracil in RNA, A=U)

• watson-crick base pairing

base stacking - double helix

Base stacking between the hydrophobic bases minimizes their contact with water and stabilizes

the double helix.

• base-stacking is a form of van der Waals forces

• bases are slightly offset so they are not directly on top of one another

• the bases lie in a plane almost perpendicular to the axis of the helix

major and minor grooves - double helix

• the offset pairing of the two strands (i.e., bases not in the center of the double strand) forms a major groove (deep) and a minor groove (shallow) on the surface of the duplex

• these grooves lie on opposite faces of the double helix and twist around the helix axis, so that if you see a major groove facing you, the minor groove is on its back side

• the base pairs are more exposed to solvent on the major groove side than minor groove side

forms of double helix

A form	B form	Z form
dsRNA DNA/RNA hybrid right handed 10.7 bp/turn	dsDNA most common right handed 10.5 bp/turn	dsDNA alternating pur/pyrim left handed 12 bp/turn

nucleotide UV light absorbtion

• 260 nm

• purines and pyrimidines are highly conjugated - resonance among rings give most of the bonds a partial double-bond character, allows UV absorption

DNA melting (denaturation) and re-annealing (renaturation)

• denaturation: DNA can be melted apart by temp or adding a chaotropic agent (urea, removing salt)

• renaturation(re-annealing): when DNA is cooled or chaotropic agents removed

how is A₂₆₀ used

• A₂₆₀ is used to distinguish between single-stranded and double-stranded DNA

• dsDNA can be disrupted by heating into ssDNA which is melted, disrupting base pairing & stacking. melting occurs at a specific temp, T_m which depends on the nucleotide sequence

• when DNA is cooled, the strands re-anneal(come back tgt), stacked bases (ds) absorb less UV light than unstacked bases (ss)

beers law

• alculating DNA/RNA concentration from A260

• Beer's law: A = εcl

  • A = absorbance (measured at 260 nm)

  • ε = molar extinction coefficient (constant for a given nucleic acid)

  • c = concentration

  • l = path length (cm, usually 1 cm for standard cuvettes)

  • Rearranged to solve for concentration: c = A / (εl)

what is T_m

• melting point

• the temp at which half the DNA is in ds form, half is in ss form

the hyperchromic shift

• The hyperchromic shift (or hyperchromic effect) is the large increase in the absorption of ultraviolet (UV) light by nucleic acids—such as DNA or RNA—when they change from a double-stranded structure to single-stranded strands. This happens during denaturation (melting) caused by heat, high pH, or chemical agents.

factors affecting T_m

• size: longer sequence length = higher T_m

• GC pairs: stacking energy is more negative(more stable) for GC than AT(3 H bonds) so more GC = higher T_m

• salt concentration: high salt concentration stabilizes the duplex, salt ions shield negatively charged phosphate on DNA backbone which can repel each other when unshield, so more salt = higher T_m