BIOCHEM501 Unit 2

basic structure of nucleotides

composed of a purine or pyrimidine base, a pentose, and a phosphate

pyrimidine

single-ring nitrogenous base; C, T, and U

purine

double-ring nitrogenous base; A and G

adenine

purine; pairs with thymine (DNA) and uracil (RNA)

guanine

purine; pairs with cytosine

cytosine

pyrimidine; pairs with guanine

thymine

pyrimidine (DNA); pairs with adenine

uracil

pyrimidine (RNA); pairs with adenine

ribose

pentose for RNA; -OH on C2

deoxyribose

pentose for DNA; no -OH on C2

glycosidic bond (N-glycosidic linkage)

the connection between the nitrogenous base and the pentose; between the 1’ position on the pentose and an N on the nitrogenous base

nucleoside

a nitrogenous base connected to a pentose

nucleotide

nucleosides with phosphoryl group(s) attached via ester linkage

nucleoside monophosphate

nucleoside with one phosphoryl group attached; what we see in the final nucleic acid polymer

nucleoside triphosphate

nucleoside with three phosphoryl groups attached; serve as high-energy building blocks used by cells to synthesize the nucleic acid polymer

phosphodiester bond

backbone of nucleic acid structure; between the 5’ and 3’ end of pentose

phosphodiester bond mechanism

1. base activation of 3’ OH

2. nucleophilic attach of 3’ OH on an “activated” 5’ phosphate (LG attached to drive rxn foreward)

cleavage of phosphodiester bonds

1. base activation of H₂O molecule

2. nucleophilic attack on phosphodiester bond

Watson-Crick-Franklin (WCF) H-bonding

occurs between bases on strands oriented anti-parallel to one another; produce double-helix

hydrogen bond donors in the bases

adenine: NH₂

cytosine: NH₂

guanine: N1H, NH₂

thymine: N3H

hydrogen bond acceptors in the bases

adenine: N1

cytosine: N3, O

guanine: O

thymine: O on C4, O on C2

WCF base pairs

• adenine forms two H bonds with thymine

• guanine forms three H bonds with cytosine

anti-anti conformation

B-form double-stranded DNA double-helix

• most common

• right-handed helix; 10.4 bp per turn

• strands are anti-parallel

• sugar-phosphate backbone on the outside

• nucleobases on the inside

• asymmetrical

base-stacking interactions (dsDNA helix)

• van der Waals interactions between hydrophobic nucleobase faces (sterics)

• π-stacking (electronic effects)

reenforce the individual A/T and C/G H-bonding interactions to stabilize the dsDNA helix

major groove

allows for sequence-specific interactions to occur from the outside without unwinding the strands

preferred RNA nucleotide conformer

C3’-endo conformer; the 2’ position of the sugar contains an OH, and the C2-endo conformation creates steric problems with the nucleobase

preferred DNA nucleotide conformer

C2’-endo conformer; the 2’ position is an H, so there are no steric issues, however, both conformations are still possible

B-form dsDNA conformation

C2’-endo conformation; places phosphates farther apart from one another

A-form dsDNA conformation

C3’-endo conformation; more tightly spaced backbone

Hoogsteen base pairs

• syn/ani conformation

• possible A/T, C/G, or G/G base pairs

• far less common than WCF

• allows 3-4 strands in a helix

• found in damaged DNA

RNA primary (1°) structure

linear sequence of nucleotides linked together by phosphodiester bonds in a single strand

RNA secondary (2°) structure

Watson-Crick and Hoogsteen pairs, formed within a single-stranded RNA molecule as it folds back on itself

RNA tertiary (3°) structure

3D arrangement of folded RNA molecule, formed by long-range interactions between 2° structure elements like stems and loops

why is DNA the primary form of information storage?

• RNA is more susceptible to degradation (2’ OH can lead to breakdown of the chain)

• easier access to information via the major groove

when does DNA need to be separated into strands?

• DNA is denatured during genome replication

• DNA melts at the 5’ end of a gene before it is expressed

which base pairs form stronger interactions?

cytosine and guanine

T_m

temperature at which the helix is half double-stranded, half single-stranded (50% denatured)

how is T_m related to dsDNA stability?

stable helix = high T_m

unstable helix = low T_m

what determines the melting temperature of dsDNA?

relative A/T and G/C content; more C/G bp make it harder to separate strands

why is knowing T_m useful?

• tells us what temperature we need to go above to cause dsDNA strands to denature (or what temp to go below to get two strands to anneal)

topology

describes the 3D shape of dsDNA; how it twists and arranges itself

Lk = Tw + Wr

linking number; cannot be changed by deforming the structure

Lk = Tw + Wr

twists; the number of times each of the curves rotates around the central axis C of the double helix

Lk = Tw + Wr

writhe; the number of times the intact B-form helix twists about itself

can topology be changed?

no; any configuration with the same Lk value is the same topography

Lk₀

• natural twist of relaxed B-form dsDNA

• equal to (# of bp) / 10.4

what determines observed topology?

for a given DNA topology Lk, the Tw will be ~Lk₀ and Wr will account for the difference between Lk and Lk₀

ΔLk = Lk - Lk₀

difference between the “natural twist” and a actual topology

ΔLk < 0

DNA is under wound and negative supercoiling will result

ΔLk > 0

DNA is overwound and positive supercoiling will result

ΔLk = 0

DNA is relaxed

DNA topoisomerase

enzymes that relieve torsional strain and prevent tangling in DNA by creating temporary single- or double-strand breaks in the sugar-phosphate backbone

type I topoisomerase mechanism

1. cut one strand of the double helix

2. attach Tyr to the DNA phosphate backbone

3. pass unbroken strand through the break, or rotate broken strand around intact one to release tension

4. seal the cut back together

type II topoisomerase mechanism

1. cut both strands of the double helix

2. pull unbroken double-stranded section of DNA through the gap

3. seal cut back together

histones

small, highly alkaline proteins found in eukaryotic and archaeal cell nuclei that package and order DNA

nucleosome

DNA + histone nucleoprotein

30nm fiber

higher-order structure of DNA and histone proteins formed by the tight folding and compaction of nucleosome arrays

histone methylation

adding methyl groups to histone proteins to bind chromatin and turn off gene transcription; makes DNA less accessible

histone acetylation

adding an acetyl group to histone proteins to relax chromatin and turn on gene transcription; makes DNA more accessible

heterochromatin proteins

bind across methylated histones to promote chromatin compaction

euchromatin

accessible; transcription can occur; marked by histone acetylation

heterochromatin

inaccessible; transcription cannot occur; marked by histone methylation and binding of heterochromatin proteins