BIOCH 200 - Nucleic Acids

Avery, MacLeod, and McCarty

Determined that DNA was Griffith's "Transforming Factor"

Hershey and Chase

Showed that DNA was the genetic material not protein through radioactive progeny

Mononucleotides

ATP, ADP, FMN

Dinucleotides

FAD, NAD+, dCdG

Structure of dAMP

Phosphate, deoxyribose and nitrogen-containing aromatic base

Structure of FMN

Phosphate, ribitol and nitrogen-containing aromatic base

2 Common Sugars

Deoxyribose (DNA) and Ribose (RNA)

Pyrimidine

1 carbon ring with 1N, 3N

Purine

2 carbon rings with four nitrogens

Which nitrogenous bases are pyrimidine?

Cytosine, thymine, uracil

Which nitrogenous bases are purine?

Adenine and Guanine

Structure of Cytosine

Structure of Thymine

Structure of Uracil

Structure of Adenine

Structure of Guanine

Where are purines and pyrimidines connected to their sugars within nucleotides?

Pyrimidine = N1

Purines = N9

Structure of a Ribose vs Deoxyribose

Ribose has as hydroxyl on carbon 2 while deoxyribose does not

Naming Purines

-ine become -osine

Naming Pyridimidines

-ine become -idine

Structure of ATP

Structure of dATP

T/F. Phosphate is usually at the 5' position

True

Ester

RCOOR

Phosphoester

Link carbons to phosphate groups

Phosphodiester

Link two different carbons to one phosphate group

How are nucleotides linked to form nucleic acids?

Nucleotides are linked by 3'-5' phosphodiester bonds

Phosphoanhydrides

Links two phosphates to each other

What directionality are nucleotide sequences written in?

5' to 3'

Primary Structure of DNA

Sequence of nucleotide residues (i.e. AGTC)

Secondary Structure of DNA

Chains of nucleotides - double helix

Is the sugar-phosphate backbone polar or non-polar?

Polar

Is the sugar-phosphate backbone in RNA or DNA more polar?

It is more polar within RNA

Nucleotide Nomenclature - Phosphates attached to a single carbon/OH groups

Mono-/di-/tri-

Nucleotide Nomenclature - Phosphates attached to multiple carbon/OH groups

Bis-/tris-

T/F. RNA is more susceptible to hydrolysis of phosphodiester bond than DNA. Why or why not?

True, the presence of the 2C' OH group makes it more susceptible (DNA is more stable)

Cytosine Deamination

Spontaneous uncatalyzed reaction that converts the NH2 to a carbonyl group forming a uracil

Dideoxynucleotide

Missing a hydroxyl group (-OH)

Properties of the Bases

Heterocyclic, aromatic, electron delocalization, planar, poorly soluble in water, largely hydrophobic

Beer-Lambert Law

Used to relate the concentration of nucleic acid to the absorbance

Erwin Chargaff (Chargaff's Rule)

Discovered that DNA composition varies, but A = T and G = C (chargaff's rule)

B-form DNA

Antiparallel strands with a right-handed twist stabilized by base stacking and hydrogen bonds

Base Stacking

Van der Waals interactions and hydrophobic forces

Complementary Sequences

Poly nucleotides are non-identical and are anti-parallel however they obey chargaff's rule

DNA Denaturation

1. Separation of DS to SS

2. Disruption of non-covalent forces (base stacking and HB)

3. Essential for some cellular processes

4. Changes in absorption properties occurs

Relative Absorbance of Double Stranded vs Single Stranded DNA

SS have a higher absorbance due to more bases (which absorb UV) exposed

Tm of AT vs GC pairs

AT pairs have a lower melting point (2HB) while GC pairs have a higher melting point (3HB & strong base stacking interactions), therefore higher GC content = higher Tm

Tm

Midpoint of melting

T/F. AT pairs tend to separate first during denaturation.

True

Why is DNA denaturation measured at 260mm?

UV is most strongly absorbed at this wavelength

Hyperchromicity

Increase in UV absorbance when DS => SS

Hypochromicity

Decrease in UV absorbance when SS => DS

T/F. Denaturation must occur for replication or transcription.

True

As the temperature increases....

DS begins to denature, hydrogen bonds break causing separation forming SS and INCREASES UV ABSORPTION

As the temperature decreases...

SS begins to anneal and form hydrogen bonds through fast zippering and DECREASES UV ABSORPTION

Renaturation

Reformation of dsDNA through proper base pairing, nucleation and zippering so it regains its native conformation

Steps of Renaturation

1. Nucleation (slow)

2. Zippering (fast)

Factors that Affect Tm

1. pH

2. Salt concentration

How does pH affect Tm?

Very low or very high destabilizes DNA, lowering the Tm

How does salt concentration affect Tm?

Salt ions shield negative charges on phosphate reducing repulsion and stabilizing the double helix

Increase salt = ___ Tm

Increase Tm

(Stabilizes double helix)

Decrease salt = ___ Tm

Decrease Tm (destabilizes double helix

T/F. Denaturation and renaturation behaviour of nucleic acids enables hybridization.

True

What determines the Tm of a hybrid?

How well they match

More similar match = ____ Tm

Higher Tm

Worse match = ____ Tm

Lower Tm

Intrastrand Base Pairing

Hydrogen bonding between bases on the same strand of nucleic acids

Interstrand Base Pairing

Hydrogen bond forming between complementary bases

T/F. The structure of RNA is LESS dependent on nucleotide sequence than double stranded DNA.

False, RNA is MORE dependent on nucleotide sequence

What type of base pairs can RNA form?

G = C (triple bond)

A = U

G = U

What is the usual max absorbance for DNA and RNA?

~260nm

What is the usual max absorbance for protein?

~280nm

What is the ideal ratio for pure DNA and RNA?

DNA = 1.95

RNA = 2.1

What does it mean when the ratios are lower than the ideal ratio?

Potential protein contamination

Order the following from highest to lowest absorption A260nm.

DS, Free nucleotides, SS

Free nucleotides > SS > DS

Dinucleotide

two nucleotides covalently bonded together by a single phosphate