Left: Two of the 115 images encoded on the gold record ...sites.fas.harvard.edu/~lsci1a/9-28notes.pdf · Life Sciences 1a Lecture Slides Set 3 ... storage device Blueprint ... nucleic

Professor David Liu and Brian Tse, Life Sciences 1a page 2

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

Left: Two of the 115 images encoded on the gold record aboard the Voyager Ispacecraft, the farthest manmade object from the sun. Right: The binary imageencoded by the November 16, 1974 radio transmission aimed at the M13 star clusterusing the radio telescope at Arecibo, Puerto Rico. Designed to convey the mostimportant elements of our collective knowledge, both messages prominently featureDNA.


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

In the last lecture we were introduced to the molecules of life and the basic components ofHIV. We learned that the core of the virus contains two strands of a nucleic acid polymer. Inthis lecture we’ll take a detailed look at nucleic acids and learn how their molecular structuresexplain their crucial biological roles.

1.The primary biological roles of nucleic acids

Nucleic acids in the form of DNA and RNA are among the key macromolecules of life. Allknown living organisms require DNA or RNA at some point during their life cycle, with the vastmajority dependent on both nucleic acids. The ubiquity of nucleic acids in forms of life asdiverse as bacteria and humans suggests that their biological roles must be very fundamental.Indeed, nucleic acids possess two unique biological features essential to life.


Nucleic Acids Encode the Molecules of Life

Nucleic acid Gene Protein

Informationstorage device Blueprint House

ATGTACGTAGCTAAGTGATCTTGACTGACGGGTACCGTGCTGATCGTGACTGATTTTCGAGGAGGATCAATCTAATAATCTAGA

First, nucleic acids carry the information encoding the molecules of life. More specifically,DNA and RNA directly encode the structure of proteins. The physical manifestation of a geneis a segment of DNA that encodes a protein. Nearly all of the molecules within living systemseither are proteins that are directly encoded by nucleic acids, or are non-protein molecules thatare generated by the actions of proteins (and therefore are indirectly encoded by nucleicacids). The precise way in which nucleic acids encode proteins will be described in detail in anupcoming lecture. For now, however, simply appreciate that nucleic acids are the blueprint forproteins, and therefore the blueprint for much of the cell. Each of the proteins that performthe duties required for HIV to infect and hijack cells is encoded in the nucleic acid moleculesthat lie in the core of each HIV virus.


Cell

Nucleus

Chromosome(Complete set = genome)

DNA

Organization of DNA in the Cell

The complete set of nucleic acid instructions in a living system is called a genome, and thegenome of an organism is typically organized into one or more chromosomes. Aside from yoursperm or egg cells, each of your cells contains 46 chromosomes. A useful analogy is that agenome is like a book, a chromosome is like a chapter within the book, and a gene is like asentence within each chapter.


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)

Second, nucleic acids are replicable. Among the millions of different molecules that havebeen created and studied in laboratories throughout the history of science, no molecules havebeen able to match the ability of DNA and RNA to replicate in an extremely efficient andaccurate manner. Nucleic acid replication either in the laboratory or in the cell can take placewith remarkable speed. By the end of this set of lectures, we will understand how it is possiblethat literally hours after a single DNA molecule begins to replicate, it is possible to createbillions of identical copies of that molecule. DNA replication relies on proteins called DNApolymerases, which in turn are encoded by DNA. In other words, cells replicate DNA usingmachines that are encoded within the cell's DNA!

Because every living cell must contain a DNA instruction set, cell division requires thecomplete replication of each dividing cell’s genome. As we will learn later in this course, thisrequirement is the basis of many anti-cancer drugs that attempt to block the division of tumorcells by blocking the process of DNA replication.


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

Scientists have learned to harness in the laboratory the unique ability of nucleic acids toreplicate. As we will learn in detail later in this lecture, a very common, inexpensive, andstraightforward technique called the Polymerase Chain Reaction (PCR) enables researchers totransform a test tube containing a single copy of DNA or RNA into a test tube containingmillions or even billions of identical copies of that molecule of nucleic acid. Among theenormous ranges of molecules known to mankind, nucleic acids remain unique in this respect.There is no known analogous method, for example, to replicate penicillin, or plastic, or sugar.

The abilities of nucleic acids to encode information and to replicate together form a verypowerful combination. This unique combination of features enables life to be propagated in amanner that preserves the features of a living system between generations. This combinationalso enables invaders of your cells such as HIV to reprogram your cells for their own needs.You may also begin to see how a change in an organism's genome can lead to either adetrimental or, more rarely, a beneficial change in that organism— the molecular basis ofevolution.


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?

Now that we have an understanding of the biological and laboratory roles of nucleic acids,we will devote the remainder of the lecture to revealing the chemical features of DNA and RNAthat enable these molecules to serve as replicable information carriers both in the cell and inlaboratory PCR experiments. Let’s start by defining the requirements for replicable informationcarriers. First, the molecule must be stable over the time scale within which the information isused. Second, the molecule must be recognized by the cellular machinery that replicates,repairs, and makes use of the information. Third, the molecule must contain multiple possiblestructures at a given location in order to carry information. Finally, the molecule must possessthe redundancy to enable error correction and to maximize the fidelity of the replicatedinformation.

We’ll begin our understanding of how DNA and RNA meet these requirements by examiningthe structural components of DNA and RNA.


The DNA Polymer: A Double Helix

+

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

2. The structural components of DNA and RNA

The primary structure of deoxyribonucleic acid (DNA)

Let’s revisit the structure of DNA in greater detail. As I mentioned earlier, DNA is a polymerconsisting of repeating monomer units. In the case of nucleic acids, these monomers arecalled nucleotides. The human genome is the entire sequence of ~3,100,000,000 DNAnucleotides within each of your cells, and the recent completion of the Human Genome Projectidentifying this sequence is a landmark scientific achievement. DNA as it exists in the cell isdouble-stranded, meaning that two DNA molecules are closely associated with each other. Intwo dimensions, you can think of double-stranded DNA as a zipper, with each half of the zipperrepresenting a molecule of DNA. In three dimensions, double-stranded DNA usually adopts itsfamous “double helix” structure.


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”

A nucleotide consists of three groups of atoms: a phosphate, a sugar, and a base. Here wewill examine each of these groups to learn how their chemical properties contribute to theability of DNA to serve as a replicable information carrier.


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

+

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

The phosphate groups of DNA link the nucleotide monomers. Even though I drew DNA in aneutral form in an earlier lecture, phosphates are actually negatively charged under conditionsresembling those in living cells (called physiological conditions). To understand whyphosphates are negatively charged in cells requires learning the key chemical concepts ofacidity and protonation states.

Thanks to the fact that your tongue perceives acidic molecules as sour, you already have anintuitive appreciation that some substances, such as lemon juice or vinegar, are more acidicthan others. For the purposes of this course, the acidity of a molecule is simply its tendency togive up protons (H+ cations). Lemon juice, vinegar, DNA, and RNA are all examples of acids.Indeed, the acidity of DNA and RNA is declared in their names.


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)

Why are some molecules more acidic than others? To answer this question requiresunderstanding a key concept in chemistry called equilibrium. Chemical equilibrium is asituation in which the concentrations of two interconverting states (A and B) are stable andtherefore do not increase or decrease. At equilibrium, the rate of A converting to B (i.e., thenumber of molecules in state A that are converted to state B per minute) is identical to the rateof B converting to A, and no net interconversion takes place. Equilibria are drawn with twostacking, opposing arrows placed between the two interconverting states.

These two states can be anything capable of interconversion, such as liquid water andwater vapor, or the starting materials and products of a chemical reaction. When evaluating amolecule’s acidity, the two relevant states are the protonated and deprotonated forms of themolecule. When an acid gives up a proton (when it is deprotonated), its covalent structurechanges slightly because one of its atoms is no longer bonded to a hydrogen atom. Thedeprotonated molecule, also called the conjugate base form of the molecule, is left with oneadditional electron that used to be a hydrogen atom’s valence electron; the hydrogen atom, inturn, becomes a free proton. Therefore, a neutral acid upon deprotonation forms a negativelycharged conjugate base (along with a proton). Likewise, a positively charged acid becomes aneutral conjugate base upon giving up a proton. At equilibrium, the concentration ofprotonated and deprotonated molecules is constant (but not necessarily equal).


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

A given equilibrium has a characteristic equilibrium constant (Keq) that reflects theconcentrations of each of the molecules involved in the interconversion when the system is atequilibrium. By definition, Keq is the ratio of [the product of the concentrations of molecules tothe right of the arrows] divided by [the product of the concentrations of molecules to the leftof the arrows] when the two states are at equilibrium. The concentrations must be in units ofmolarity (M). Molarity is defined as moles of dissolved molecules per liter of solution (whereone mole = 6.02 x 1023 molecules, the number of molecules that are needed to consitutute amolecule’s molecular weight in grams). Concentrations expressed in units of molarity aresymbolized by bracketed variables such as [A]. As we will explore in more detail later in thiscourse, Keq is ultimately dependent on the relative stabilities of the left and right sides of anequilibrium.

Note that you can write an equilibrium in either of two orientations (A on the left and B onthe right, or A on the right and B on the left), and the values of Keq for these two equilibriumconstants are always reciprocally related (i.e., Keq,forward = 1 ÷ Keq,reverse).

All states capable of interconversion will eventually reach equilibrium if left unperturbed,even though the equilibrium might favor one side or another (i.e., the value of Keq might be> 1 or might be < 1).


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

When the left side of the equilibrium consists of an acid, and the right side of theequilibrium consists of a conjugate base plus a proton, Keq is also equal to Ka, or the acidityconstant. In water, free protons do not really exist and instead rapidly protonate H2O to yieldH3O

+. Therefore, a more accurate equilibrium in water is Keq = ([H3O+][A–])÷([HA][H2O]); in

this case Ka is defined as Keq[H2O], which equals [H3O+][A–]÷[HA]. For convenience, the

acidity of a molecule is usually indicated by its pKa value rather than by its Ka value. Ka andpKa are related by the simple equation pKa = –log(Ka). Molecules with lower pKas are moreacidic than molecules with higher pKas. By convention, molecules with pKa values less than 7are defined as acidic in water, while molecules with pKas greater than 7 are considered basic inwater. The pKa values of several representative molecules are shown here.

From the above you can deduce that a molecule is more acidic if its deprotonated form isparticularly stable— i.e., if at equilibrium its deprotonated state is more prevalent. Moleculeswith very stable deprotonated forms are highly acidic; conversely, molecules with unstableconjugate base forms are not acidic and are referred to as basic instead. One key factor thatimproves the stability of the deprotonated form (and therefore increases the acidity of amolecule) is the presence of electronegative atoms or groups that are positioned very close tothe electrons involved in bonding the proton that is being released.


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)

The combination of acidic or basic molecules dissolved in an aqueous solution imparts anacidity or basicity in that solution. The acidity or basicity of a solution is described by anumber called its pH. By definition, the pH of a solution is equal to –log [H+]. Since in waterprotons actually become H3O

+ cations, pH = –log [H3O+] in water. Therefore, a solution

containing a proton concentration of 0.1 M has a pH of 1. As you can deduce, the greater theconcentration of protons in solution, the lower the pH of that solution. Neutral aqueoussolutions have a pH of 7.0. Acidic solutions such as lemon juice have a pH below 7, whilebasic solutions such as ammonia-containing window cleaners have a pH above 7.


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)

The protonation state of a molecule depends on both its pKa as well as the pH of thesurrounding solution. Under physiological (pH = ~7) conditions, a molecule with a pKa of 7exists as an equal mixture of protonated and deprotonated forms. Molecules with pKas lessthan 7 are mostly deprotonated and adopt their conjugate base forms in pH 7 solutions.Conversely, molecules with pKas above 7 are mostly protonated under physiological conditions.

The relationship between pKa, pH, and protonation state is given by the Henderson-Hasselbalch equation: pKa = pH + log ([HA]/[A–]). The Henderson-Hasselbalch equation isvery useful for understanding the molecules of life because it allows you to determine whatfraction of a molecule exists in a protonated versus deprotonated form at any given pH. Frominspecting this equation, you can deduce the two crucial results that (i) increasing the pH of asolution by one pH unit increases the fraction of deprotonated molecules by exactly 10-fold,and (ii) when pH = pKa, the concentrations of protonated and deprotonated forms are equal.


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

For example, because acetic acid (the acidic molecule in vinegar) has a pKa of 5, only onemolecule out of 100 is protonated in aqueous pH 7 solutions; the other 99% of the acetic acidmolecules have given up their acidic protons. On the basic side of the pKa spectrum,ammonium cation (+NH4) with a pKa of 9 is 99% protonated in pH 7 solutions because 7 is twounits lower than its pKa. If you were to raise the pH of the solution to 8, however, the fractionof the ammonium cations that are protonated would fall to 90%.


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

As a dramatic example of the importance of understanding the relationship between pH,pKa, and protonation state, consider the drug shown here named Prilosec. Prilosec is used totreat heartburn; annual sales hover around $5 billion, which makes Prilosec among the mostwidely used drugs on the market.

A key feature of prilosec is that it can exist in either a protonated or deprotonated form.The pKa of this crucial protonation/deprotonation is 4. These two forms of Prilosec have verydifferent properties. The deprotonated form is inactive but readily travels between cells. Theprotonated form is active but cannot move between cells.

Based on your understanding of pH, pKa, and protonation state, you can understand thesecret of Prilosec’s success. The drug travels between cells in your body until the pH of theenvironment is lower than 4. At that point, the majority of Prilosec molecules becomeprotonated and biologically active, and no longer leave that acidic environment. The net resultis that Prilosec moves freely in an inactive state throughout your body until it stumbles uponthe acidic environment of the stomach lumen (pH 1). In this acidic environment, Prilosec is99.9% protonated, and therefore is both trapped in its target space and is also predominantlyin its active form.


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

You can now also understand how the acidity of the protonated phosphate group (pKa =~2) causes the entire backbone of a strand of DNA to be decorated with negative charges atneutral pH. These negative charges usually form ionic bonds with positively charged ions suchas sodium ions, and in cells are often also associated with positively charged proteins. Theformation of ionic bonds between positively charged protein building blocks that containgroups with pKa values > 7, such as arginine and lysine (which we will discuss in a couplelectures), and negatively charged phosphate groups is one of the primary mechanisms bywhich living systems grab onto and manipulate DNA.

In extremely pure water, you may be surprised to learn that DNA no longer is capable offorming double-stranded structures. The reason is that the multiple negatively chargedphosphate groups in each strand of DNA repel each other as two complementary strandsapproach each other. This electrostatic repulsion is so great that it cannot be overcome by thefavorable hydrophobic and base stacking interactions that occur upon formation of double-stranded DNA. Cations such as sodium and magnesium therefore play a crucial role in DNAhybridization by neutralzing their negative charge. Only when enough cations are available toneutralize a sufficient number of phosphate negative charges can DNA form a double helix.


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

The negative charge of phosphate groups is also crucial for the stability of DNA. As we willlearn in detail during a later lecture, DNA carries the information encoding life. To fulfill thiscrucial role requires that DNA be extremely resistant to common ways by which moleculesspontaneously degrade. One of the most common ways that the molecules of life degrade inwater is through a process called hydrolysis (literally, “water rupture”). In the case of DNA,hydrolysis results in breakage of the DNA backbone. Hydrolysis of both strands of a double-stranded DNA containing necessary information for life leads to cell death.

The first step in DNA hydrolysis is bringing the non-bonded electron pairs on the oxygenatom of water into very close proximity of one of the phosphorus atoms in the DNA backbone.As we have previously seen, electrons repel each other. When the non-bonding electron pairsof an oxygen atom approach the phosphorus atom in a phosphate group, they are stronglyrepelled by the negative charges surrounding the phosphate oxygens. In other words, theabundance of electron density on the phosphate oxygens effectively shields the phosphorusatom from being attacked by water. Indeed, when DNA is chemically modified so as toremove the phosphate negative charges, it becomes much more prone to hydrolysis.

You may conclude from examining the structure of a phosphate group in DNA that thegroup is chiral; after all, the phosphorus atom is bonded to what appear to be four differentgroups (=O, –O–, and two different –O–ribose groups). However, the two oxygen atomgroups in a phosphate are actually identical, with the negative charge residing on each oxygenatom about 50% of the time. This concept, called resonance, will be described in detail in anupcoming lecture. Because two of the four groups bonded to the phosphorus atom areidentical, the phosphate groups in DNA are not chiral.


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

The sugar group in DNA; strand orientation and macromolecular chirality

The sugar group in DNA serves as a scaffold to hold the phosphates and bases in theirproper positions. In DNA, this sugar is actually a variant of ribose called deoxyribose. Each ofthe five carbon atoms in ribose or deoxyribose are numbered 1' to 5' (the prime designation isused to specify that the numbering refers to the sugar and not to other parts of a nucleic acid).You'll notice that the phosphate groups are attached to DNA at the 5' carbon and at the 3'carbon of deoxyribose.


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

These attachment points give each DNA strand an orientation. If you start at the middle ofa ribose ring and trace a DNA strand in the direction of the nearest 5' carbon (also called the 5'direction), you will reach the 5' end of the strand. Likewise, if you trace a DNA strand from themiddle of a ribose ring in the direction of the nearest 3' carbon, you will reach the 3' end of thestrand. If you reexamine the structure of double-stranded DNA, you will see that the twostrands are oriented in opposite directions, such that the 5' end of one strand lies closest tothe 3' strand of the other strand of the double helix. This orientation is called 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)

Ribose and deoxyribose sugars are both chiral molecules that exist in enantiomeric formscalled D-(deoxy)ribose and L-(deoxy)ribose. Only the D-deoxyribose enantiomer is found inDNA. As is often the case with the chiral molecules of life, the non-natural enantiomer ofdeoxyribose is not recognized by the cell as a component of DNA.


The Chirality of Ribose Determines theMacromolecular Chirality of DNA

D-riboseRight-handeddouble helix

Wrong

WrongWrong

Wrong

Wrong

Double-stranded DNA almost always adopts a three-dimensional structure containing a“right-handed” twist, meaning that if you curl the fingers of your right hand along the groovesof the DNA double helix, your right hand moves in the direction of your extended thumb.DNA’s natural right-handed twist is often drawn incorrectly in popular magazines (andoccasionally in scientific journals as well!). Even ignoring the detailed arrangement of thechemical bonds in DNA, this right-handed twist cannot be superimposed on its mirror image.This chiral feature of the overall macromolecule is an example of macromolecular chirality.Macromolecular chirality is ultimately derived from the chiral groups within a macromolecule.Because deoxyribose is the only chiral group in DNA, we can deduce that the right-handednessof the twist of double-helical DNA ultimately arises from the presence of D-deoxyribose.Indeed, laboratory-created double-stranded DNA containing L-deoxyribose would adopt a left-handed twist.


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

The bases of DNA

The phosphate and deoxyribose groups of DNA do not vary between different DNAmolecules and cannot serve as information carriers. One type of efficient information carrierencodes information based on the order in which a collection of different information-encoding“bits” exists in a string of these bits. In the case of DNA, these information-encoding bits arethe four bases of DNA.

The bases of DNA are planar cyclic structures containing carbon, nitrogen, oxygen, andhydrogen atoms. Rings comprising both carbon and non-carbon atoms (such as the four DNAbases) are called heterocycles. The four DNA bases are called adenine, cytosine, guanine, andthymine but are usually referred to as A, C, G, and T respectively. These four bases whenconnected to a ribose or deoxyribose sugar are called the nucleosides adenosine, cytidine,guanosine, and thymidine. The two larger bases, A and G, are collectively classified aspurines, while the two smaller bases, C and T, are pyrimidines. Because the DNA bases areplanar, they are by definition superimposable with their mirror images and therefore are notchiral.


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

Although A, C, G, and T differ in structure, they share some important common features. Inaddition to being planar heterocycles, all four bases contain a nitrogen atom that in DNA iscovalently bonded with the 1’ carbon atom of deoxyribose. Also, the four DNA bases eachcontain a unique constellation of hydrogen bond donors and acceptors. The donors andacceptors are precisely arranged such that A can form two hydrogen bonds with T, and C canform three hydrogen bonds with G at every nucleotide position within double-stranded DNA.James Watson and Francis Crick are credited with first deducing that A pairs with T and G pairswith C in the manner shown here. Watson and Crick apparently knew this discovery wasimportant, as they walked into the Eagle Pub in Cambridge, England on February 28, 1953 andannounced to the pub patrons “We have found the secret of life!”. Their seminal 1953discovery was recognized with the 1962 Nobel Prize in Physiology or Medicine, awarded toWatson, Crick, and their collaborator Maurice Wilkins.


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

The fact that the individual strands of double-stranded DNA form a string of Watson-Crickbase pairs means that the two DNA molecules in a double helix are complementary. In otherwords, you can deduce the exact sequence of nucleotide bases in either strand if you are giventhe sequence of the opposite strand simply by pairing A with T, C with G, G with C, and T withA. The complementary nature of double-stranded DNA is crucial for a number of fundamentalprocesses in all living systems. For example, DNA’s complementarity creates a way of backingup the information stored in a double helix of DNA. If any one DNA strand is damaged, thecell can use the complementary strand to infer the correct sequence of bases in the damagedstrand and repair the damage. More details of this crucial and elegant DNA repair strategy willbe presented later in this course.


• 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

As a second example of a fundamental biological process that takes advantage of DNA’scomplementarity, each strand of a cell’s double-stranded DNA is used to infer the sequence ofthe opposite strand during cell division. In fact when a cell divides, each of the two daughtercells contains one of the original DNA strands of the parental cell. This mode of DNAreplication is called semi-conservative replication and will be revisited later in this section of thecourse.


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

The primary structure of ribonucleic acid (RNA)

Ribonucleic acid, or RNA, is identical to DNA in its basic molecular structure with twoimportant exceptions. First, as its name implies, RNA uses ribose rather than deoxyribose asits sugar scaffold and therefore contains a 2’ hydroxyl group where DNA contains only a 2’ –Hgroup. The second basic structural difference between RNA and DNA lies in one of the fourbases. Instead of thymine (T), RNA uses uracil (U). The structure of thymine and uracil arevery similar. Thymine has a –CH3 group (a methyl group) connected to one carbon of thepyrimidine ring, whereas uracil has a –H group at this position. Cells make thymine by addinga methyl group to uracil.


RNA Exhibits Great Structural Diversity

Tetrahymena rRNAtRNA

Hammerhead RNAHepatitus deltavirus RNA

Scientists hypothesize that the 2’ hydroxyl group allows RNA to fold more easily intocomplex three-dimensional shapes such as the structures shown here compared with DNA,possibly because the 2’ hydroxyl group provides an extra hydrogen bond donor and acceptorcompared with DNA. The ability of RNA to fold into complex shapes is crucial for its proposedrole in the origins of life, as will be discussed later in this course.


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

Why does DNA use deoxyribose instead of ribose?

Let’s take a closer look at the consequences of one of the molecular differences betweenDNA and RNA. Cells make deoxyribose by replacing the –OH group (also called a hydroxylgroup) at the 2’ carbon of ribose with a –H group. This seemingly simple transformationactually involves sophisticated chemistry that has proven thus far to be impossible to achievein one step in the laboratory using purely manmade molecules. Why does nature bother toremove the hydroxyl group from the 2’ carbon atom of ribose when constructing DNA? It isimpossible to answer questions about ancient chemical origins (also called chemical etiologyquestions) with certainty, but a likely explanation is that the removal of the 2’ hydroxyl groupof ribose dramatically increases the stability of DNA. The non-bonded electron pairs on theoxygen atom of this 2’ hydroxyl group are positioned close to the phosphorus atom of thenearby phosphate group. Just as the approach of the oxygen atom in water to thisphosphorus atom can lead to the hydrolysis of DNA, the approach of the ribose 2’ oxygen atomto this phosphorus atom can also lead to RNA cleavage.

Even though the negative charge surrounding the phosphate group helps to protect thephosphorus atom from attacking water molecules, the 2’ oxygen atom of ribose is a particularlyserious threat. Unlike the oxygen atom of a water molecule, the 2’ oxygen atom is alwayspositioned very close to the phosphorus atom and does not need to rely on a chanceencounter between a water molecule and a phosphorus atom. Chemical reactions that requiretwo molecules to randomly bump into each other (such as DNA hydrolysis) are calledintermolecular reactions, while reactions that occur between groups within the same molecule(such as RNA cleavage involving the 2’ ribose hydroxyl group) are called intramolecularreactions. Frequently intramolecular reactions take place much faster than intermolecularreactions because of the pre-organized nature of the former. As we will learn in a later lecture,this pre-organization is a major way that biological macromolecules guide chemical reactions.

Documents

Left: Two of the 115 images encoded on the gold record ...sites.fas.harvard.edu/~lsci1a/9-28notes.pdf · Life Sciences 1a Lecture Slides Set 3 ... storage device Blueprint ... nucleic