1

Life Sciences 1aLecture Slides Set 3Fall 2006-2007Prof. David R. Liu

Lectures 3-5: Nucleic acids & the chemical requirements for replicating information

1. The primary biological roles of nucleic acids

2. The molecular components of DNA and RNA

a. The primary structure of deoxyribonucleic acid

b. The phosphate group in DNA; equilibrium, acidity, and protonation states

c. The sugar group in DNA; strand orientation and macromolecular chirality

d. The bases of DNA

e. The primary structure of ribonucleic acid

f. Why does DNA use deoxyribose? Why T?

3. The factors behind DNA base pairing

a. DNA hybridization as an equilibrium

b. The role of hydrogen bonding

c. The role of the hydrophobic effect and base stacking

4. The molecular basis of DNA replication

a. DNA replication; chemical reactions, substrates, and products

b. The role of DNA polymerase: faster and more accurate DNA replication

c. The polymerase chain reaction (PCR) and its impact on the life sciences

Required: Lecture Notes,McMurray 796-813, 197-202,Ch. 10, 837-838; Alberts pp. 56-58, 68-69, 76-77, 168-171,175-177, 195-197

Lecture Readings

2

Nucleic Acids Encode the Molecules of Life

Nucleic acid Gene Protein

Informationstorage device Blueprint House

ATGTACGTAGCTAAGTGATCTTGACTGACGGGTACCGTGCTGATCGTGACTGATTTTCGAGGAGGATCAATCTAATAATCTAGA

Cell

Nucleus

Chromosome(Complete set = genome)

DNA

Organization of DNA in the Cell

3

Nucleic Acids are ReplicableInformation Carriers in the Cell

DNA replication

• Each cell division requires replication of the cell’s genome

cell replication

(very complex)

(understood inmolecular detail)

Molecular Replication in the Laboratory:The Polymerase Chain Reaction (PCR)

one moleculeof penicillin

multiple copies of thatpenicillin molecule

N

O

NH SH

O

OH

OPhN

O

NH SH

O

OH

OPh

N

O

NH SH

O

OH

OPh

N

O

NH SH

O

OH

OPh

N

O

NH SH

O

OH

OPh

N

O

NH SH

O

OH

OPh

N

O

NH SH

O

OH

OPh

N

O

NH SH

O

OH

OPh

N

O

NH SH

O

OH

OPh

N

O

NH SH

O

OH

OPh

N

O

NH SH

O

OH

OPh

no knownprocess

X

one molecule of DNA

$2 of readilyavailable

ingredients

1 hourPCR 1,000,000 copies of

that molecule of DNA

• The ability to replicate (both in the cell and in thelaboratory) is a unique feature of nucleic acids

4

What are the Requirements for aReplicable Information Carrier?

1) Resist degradation2) Be recognized by cellular machinery3) Contain multiple possible structures (bits) at each position4) Possess redundancy for error correction and faithful copying

How does DNA (or RNA) satisfy theserequirements?

The DNA Polymer: A Double Helix

+

• Double-stranded DNA typically adopts a double-helicalconformation; single-stranded DNA is more disordered

5

The DNA Monomer: A Nucleotide

OO N

N

N

NH2

O

NP

OO

O

Base

Sugar

Phosphate

• The monomer of nucleic acids is the nucleotide, whichconsists of a phosphate, sugar, and “base”

The Phosphate Backbone, Acids,and Conjugate Bases

acid(protonated form)

conjugate base(deprotonated

form of the acid)

O

P

O

O

OH

O

P

O

O

O-H+

+

proton

• Acidity is the tendency of a molecule to give up protons• The phosphate group is acidic and therefore is mostly

negatively charged under physiological conditions

+

6

Equilibrium: A Dynamic Balancing Act

Liquid water Water vaporOH

H

O

H

H

O

HH

OH

H

OH H

O

H

H

O

H

HO

H

H

O

H

H

O

H

H

C

O

O

+Water,

carbon dioxide OC

O

OHH

Carbonic acid

+Single-strandedDNA

Double-strandedDNA

• At equilibrium, the concentrations of two interconvertingstates do not change (forward rate = reverse rate)

The Equilibrium Constant (Keq)

A B C D+ +

Keq (reverse) = Keq (forward)1=

Alternatively: A B+C D+

[A] [B][C] [D]

Keq = [A] [B][C] [D]

at equilibrium[A] = concentration of A in moles per liter1 mole = ~6 x 1023 molecules

• Keq reflects which side of an equilibrium is favored(> 1: right side; < 1: left side), and to what degree

7

Acidity and pKa

HO OH

OO OH

OO

H+

+ HO OH

OO OH

OO-H

O

OH

O

O-H+

+

O

P

O

OH

OHH

O

P

O

OH

OH-H+

+

O

P

O

O

OH

O

P

O

O

O-H+

+

H+

+ HN

HHH

N

HH

H+

acid conjugate base

Phosphoric acid

DNA backbone

Citric acid

Acetic acid

Ammonium ion

Ka = Keq for AH        A– + H+ (the deprotonation reaction)(in water, H+ becomes H3O+ and Ka = Keq[H2O] = [A–][H3O+]/[HA])

2

2

3

5

9

pKa (= –log Ka)

Incre

asin

g A

cid

ity

10-2

Ka

10-2

10-3

10-5

10-9

What Does pH Mean?

AH A– H++

pH = –log [H+]

• The lower the pH, the higher the[H+], indicating a more acidic solution

• Each pH unit represents a 10-foldchange in [H+]

(In water, pH = –log [H3O+])

Keq (= Ka)

8

The Relationship Between pKa, pH,and Protonation State

pKa = pH + log [HA][A–]

Two key implications:

1) If pH increases by 1: The ratio of [A–] (deprotonated) to [HA] increases by 10-fold

Conversely, if pH decreases by 1: The ratio of [HA] (protonated) to [A–] increases by 10-fold

2) When pH = pKa, then [A–] = [HA]

(aka the Henderson-Hasselbalch equation)

Examples: pKa, pH, and Protonation StatesO

OH

O

O-H+

+ pKa = 5Acetic acid

At pH 5.0 1 : 1At pH 7.0 1 : 100At pH 9.0 1 : 10,000

H+

+ HN

HHH

N

HH

H+

pKa = 9Ammoniumcation

At pH 5.0 10,000 : 1At pH 7.0 100 : 1At pH 9.0 1 : 1

9

Prilosec, pH, and pKa

OH3C N

HN

S

O

N

H3C O

CH3

CH3

H

OH3C N

HN

S

O

N

H3C O

CH3

CH3

H++

Prilosec (omeprazole, sold by AstraZeneca)Treats heartburn; 1998-2002 sales averaged $5,000,000,000 per year

• Target of prilosec: a protein in the acid-secreting parietalcells of the stomach, facing the stomach lumen (pH = ~1)

• pH 7 (typical cells): prilosec is 99.9% inactive and mobile• pH 1 (stomach lumen): prilosec is 99.9% active and immobile

pKa = 4

Accumulates in cellsBiologically active

Travels between cellsBiologically inactive

Phosphates Form Ionic Bonds With Cations

O

P

OO

O

–99.999% in thedeprotonated,anionic form at pH 7

OBase

O

P

OBase

O

O

O

O

-HN

NH

O

Protein DNA

+

Arginineside chain

N

N

H

H

H

H

• Cells form ionic bonds with the phosphates of DNA andRNA to recognize and manipulate nucleic acids

pKa = 12

O

P

OO

HO

H+ +pKa = 2

10

The Phosphate Group Shields DNA

OB

O

P

OB

O

HO

O-O

O-Hydrolysis DNA strand

breakage,possiblemutation orcell death

Electrostaticrepulsion slows

down DNAhydrolysisO

P

OO

O—

HO

H

!-

• The negative charge surrounding the phosphate groupprotects DNA from hydrolysis

OB

O

P

OB

O

O

O-

OHO

H

OHO OH

HO

1'

2'3'

4'5'

(D)-2' Deoxyribosefound in DNA

OHO OH

HO OH

1'

2'3'

4'5'

(D)-Ribosefound in RNA

OO Base

O

P

OO

O 4'

3' 2'

1'5'

Ribose: The Sugar of Nucleic Acids

5’ end

3’ end

11

Ribose Structure Defines theDirectionality of DNA Strands

5'

O

O

PO

O

O

Base

O

Base

O

O

P O

O

O

O

5'

3'

3'

5'

3'5'

3'

• Double-stranded DNA is antiparallel

Ribose is a Chiral Molecule

OHO

OHHO

OHO OH

HO OH

HO

Mirror images

D-ribose(natural enantiomer)

L-ribose(not present in any natural

nucleic acids)

12

The Chirality of Ribose Determines theMacromolecular Chirality of DNA

D-ribose Right-handeddouble helix

Wrong

WrongWrong

Wrong

Wrong

The Nucleic Acid Bases

N

N

N

N

NH H

H

Adenine

N

N

N

N

O

N

H

H

HH

Guanine

Purines Pyrimidines

N

NH3C

O

O

H

H

Thymine(DNA only)

N

N

N

O

H H

H

Cytosine

• The order of basesin DNA and RNAencode information(2 bits per base)

• The bases of DNAand RNA are flat(and therefore areachiral)

• Know thyself: learnthese structures

13

Watson-Crick Base Pairing

O

O

PO

O

O

N

N

N

N

N

O

H

H

N

N

CH3O

O

H

O

O

P O

O

O

O

adenosineA

thymidineT

O

O

PO

O

O

N

N

N

N

O

ON

H

H

HO

O

P O

O

O

NO

N

N

O

H

H

cytidineC

guanosineG

• Each base displays a unique constellation of hydrogenbond donors and acceptors that can pair when juxtaposed

The Complementarity of DNAEnables Error Correction

A - T

T - A

G - C

G - C

T - A

C - G

A - T

G - C

damage

A - T

T - A

G - C

G - ?

T - A

C - G

A - T

G - C

Infer correct basesince A pairs with Tand G pairs with C

repair

=

A - T

T - A

G - C

G - C

T - A

C - G

A - T

G - C

5'

5'

3'

3'

A - T

T - A

G - C

G - C

T - A

C - G

A - T

G - C

14

• Semi-conservative replication facilitates error correction byallowing cells to distinguish new and original DNA strands

DNA Replicates Semi-Conservatively

One strand ofparental DNA per

daughter cell

Parental cell

RNA Structure

N

NH3C

O

O

H

H

Thymidine(DNA only)

N

N

O

O

H

H

Uracil(RNA only)

No methylgroup

OHO OH

HO

1'

2'3'

4'5'

(D)-2' Deoxyribosefound in DNA

OHO OH

HO OH

1'

2'3'

4'5'

(D)-Ribosefound in RNA

• RNA and DNA structure differ in two ways

15

RNA Exhibits Great Structural Diversity

Tetrahymena rRNAtRNA

Hammerhead RNAHepatitus deltavirus RNA

Why Does DNA Use Deoxyribose?

• Intramolecular reactions are much faster than correspondingintermolecular reactions

• DNA is more resistant to strand cleavage due to deoxyribose

Intermolecularreaction

slower

OB

O

P

OB

O

HO

O-O

O-

A B+Slower

DNAO

B

O

P

OB

O

O

O-

OHO

H

Intramolecularreaction

faster

OB

O

P

OB

O

HO

O-O

O

OH

A B

Faster

RNAO B

O

P

O B

O

O

O-

O

OH

OH