3 Secondary structure of DNA

what are primary structures in sequences stabilized by

1o structure is the sequence of nucleotide residues

• Stabilized by phosphodiester bonds - major one

what are higher order structures formed by and what does DNA form

• Higher order structures are formed by chains of nucleotides

• DNA forms a double helix - Each chain is connected through hydrogen bonds between the bases

what did erwin chargaff do

• Base composition varies from organism to organism

• Helped Watson and Crick deduce the double helical structure of DNA

• Chargaff’s rule is only true for dsDNA

• number of purines = number of pyrimidines

what are some properties of H bonds

• Polar interactions - electrostatic interactions

• Hydrogen atom in a H-bond is shared by two electronegative atoms - Nitrogen, Oxygen, Sulphur

• Much weaker than covalent bonds - help stabilize the structure because of their large numbers, fewer number of ionic

• stronger than dd forces and has partial overlap of electrons

what to H bonds need to form between marcomoelcules

For H-bonds to form between or within biochemical macromolecules they must be shielded from water

Note: When groups hydrogen bonded to one another they are less polar - less likely to H bond with water

where do the hbonds form in DNA

• between nucleotides

The amino and carbonyl functional groups on the four bases found in DNA allow for specific hydrogen bonding interactions between the bases.

Not all parts of the bases that can hydrogen bond are involved in Watson-Crick base pairing, other groups can h bond with proteins when they interact

what are the different forms of DNA

B form - most common, always assume its this one, secondary structure, strands are anti parallel with right handed twist, has major groove where proteins interact with it and sometimes in minor

A form - dehydrated so theres less water, right handed twist, shorter

Z form - very rare, left handed twist

what is DNA stabilized by

Stabilised by base stacking interactions and hydrogen bonds

Base stacking interactions - you dont get this with proteins and very specific nucleic acids

• Major stabilising force of secondary structure - double stranded is more stable than single

• van der Waals

• hydrophobic forces - so when you stack them you get more hydrophobic forces

what parts of DNA are polar

• Hydrophobic core/polar exterior

• Bases largely excluded from H2O - H-bonds in pairing interactions, Stabilizes H-bonds

• Ribose/deoxyribose and phosphates exposed to H2O

what are the complementary strands in DNA

# A’s in one strand = # T’s in complementary strand

# G’s in one strand = # C’s in complementary strand

• they can H bond with each other, the two strands are not identical they are complementary (watson and crick base pairing)

Complementarity involves the ability to H-bond when the strands are antiparallel.

what does chargaffs rule not apply to

doesnt apply to SS DNA or RNA

if a 28 base pair of double stranded DNA and 7 adenine how many guanine resides?

21

why do nucleic acids have a sense of direction

• covalent bonds are always formed in the same direction because the ends are geometrically/structurally distinct from each other, not identical

Is the dinucleotide pU-p-A the same as the dinucleotide pA-p-U?

no

what are the alkaline hydrolysis products of this dinucleotide

how do you name the phosphates in a nucleotide

• Phosphates attached to a single carbon/OH groups are typically identified using mono- /di-/tri- prefixes

• Phosphates attached to multiple carbon/OH groups are described using bis-/tris- prefixes

what is the absorption of free nucleotides in relation to DNA

A260nm of free nucleotides > ssDNA > dsDNA

what is DNA melting

• goes from DS to two single stranded

• starts around 50 degrees, increases then plateaus at the top

• relative absorption increases which means it starts separating

• H-bonds and base-stacking are weak forces

• Absorbance changes as DNA “melts” – base stacking changes

what is Tm

• midpoint of melting

• characteristic of the base composition, diff bases affect how much heat you need

• an index of the thermal stability of a nucleic acid

• Dependent on many conditions which include but are not limited to

  • Base number - The longer the nucleic acid strands the greater the Tm

  • Base composition and sequence

  • Solvent conditions - Salt, pH

what are DNA melting curves and the different shifts that can happen

• DNA melting curves can be measured because single stranded DNA has a higher absorbance than double- stranded.

• Hyperchromic shift - Shift from low to high absorbance, DS to SS

• Hypochromic shift - Shift from high to low absorbance, SS to DS

what is hyper/hypochromicity

• “Hyperchromicity” is a (relatively) high absorbance, SS DNA

• “Hypochromicity” is a (relatively) low absorbance, DS DNA

what bonds do you break when you denature DNA

• Denaturation = strands separate (no covalent bonds broken) - breaking H bonds between bases and base stacking

• Degradation = DNA is cut apart (covalent backbone bonds broken) - phosphodiester bonds are broken

how can you renature DNA

• slower than denaturation, 2 components slow and fast

• slow - depends on the complexity of DNA and involves nucleation, trying to line up 2 ends correctly, the more complicated the slower, drop temp slowly

• fast - zippering, regain native conformation (proper base pairing)

• you can never renature genomic DNA after you denature it

what does renaturation require and involve

• Reformation of dsDNA so it regains its native conformation

• requires proper base pairing

• involves nucleation and zippering

• heat it to break and cool it slowly to nucleate properly, if not you have to re heat it and re cool it so it goes back

degradation vs denaturation

degradation - alkaline hydrolysis, breakdown of biomolecules into smaller pieces usually irriversable, covalent bonds broken

denaturation - losing 3D shape without changing its primary chemical sequence, dna strands separating, reversible

• both can happen to DNA

what does the melting curves look like with different base compositions

GC base pairs have more base stacking interactions which make it more stable, more surface area gives more interaction

The most stable is GC, least stable is AT, as you increase GC the tm increases

• Tm is linearly related to GC content of DNA, higher Tm = higher stability

what regions of DNA are rich in AT base pairs

• DNA starts separating, genomic DNA, these regions have more AT base pairs transport

• transcription initiation bubble and DNA replication origins are often AT rich

what is pKa

An index used to express the strength of an (weak) acid

The smaller the pKa value, the stronger the acid

• refers to molecule in solutions

what is pH

An index used to express the [H+] in solution

pH measures the acidity of a given solution

lower pH = more hydrogen ions

what are 3 rules to apply to pH and pKa

At a pH below the pKa, the acid exists predominately as AH Protonated form/acid - lots of H in it

At a pH above the pKa, the acid exists predominately as A- Deprotonated form/conjugate base - easy to offload H

When the pH of the solution = pKa of the group [AH] = [A- ] i.e. exactly half dissociated

• see slides 87 - 88

what are the different pKa values for phosphate groups

pKa values of the –OH groups will likely change if the phosphate is part of a molecule.

You can have the same functional group but depending on the structure of the molecule the pKa varies

• see slide 90 - 91

what effect does pH have on denaturing

Changing pH will affect Tm - Affects protonation state of DNA and ability to H-bond, pH > 10 certain groups are deprotonated

• higher pH = lower Tm

what effect does pH have on salt concentration

• Changing salt concentrations - Ions shield negative charges on phosphate backbone

• add these to reduce negative charges

  • Mg2+

  • Positively charged proteins (histones)

• Low salt decreases Tm - Destabilizes double helix

• Increased salt elevates Tm - Stabilizes double helix

what are the conditions to denature and hybridize

Conditions to denature? Increase temp, decrease salt, increase Ph which allows

strand to separate easily

Conditions to hybridize? Low temp, high salt, low pH to around 7

• The method was used to identify similar sequences in different species

what are properties of RNA

• A pairs with U

• Often single-stranded but not always

• Like DNA, stabilised by hydrogen bonding and base stacking interactions, smaller than dna, most important to stabilize

• Intrastrand base pairing - base pairing with bases in the same strand

• DNA also has Interstrand base pairing

what can complementary RNA form

• can form double stranded helix

• not the same structure as B-DNA bc of the 2’ hydroxyl group

• right handed, stabilized primarily by base stacking interactions

what does the melting curve for RNA look like compared to DNA

Tm is defined as temperature of

melting or, more accurately, as

temperature of mid-transition.

• see slide 100