174

340

Chapter 28: Nucleosides, Nucleotides, and Nucleic Acids.Nucleic acids are the third class of biopolymers (polysaccharides

and proteins being the others)Two major classes of nucleic acids

deoxyribonucleic acid (DNA): carrier of genetic informationribonucleic acid (RNA): an intermediate in the expression

of genetic information and other diverse rolesThe Central Dogma (F. Crick):

DNA mRNA Protein (genome) (transcriptome) (proteome)

The monomeric units for nucleic acids are nucleotidesNucleotides are made up of three structural subunits

1. Sugar: ribose in RNA, 2-deoxyribose in DNA2. Heterocyclic base3. Phosphate

341

sugar base

sugar base

phosphate

sugar base

phosphate

sugar base

phosphate

sugar base

phosphate

nucleoside nucleotides

nucleic acids

Nucleoside, nucleotides and nucleic acids

The chemical linkage between monomer units in nucleic acidsis a phosphodiester

175

342

28.1: Pyrimidines and Purines. The heterocyclic base; there are five common bases for nucleic acids (Table 28.1, p. 1166). Note that G, T and U exist in the keto form (and not the enol form found in phenols)

N

NH

N

N

N

N

purine

pyrimidine

N

NH

N

NN

NH

N

NH

NH2 O

NH2

NH

N

O

NH2

NH

NH

O

O

NH

NH

O

O

H3C

adenine (A)DNA/RNA

guanine (G)DNA/RNA

cytosine (C)DNA/RNA

thymine (T)DNA

uracil (U)RNA

1

2

34

5

67

8

9

1

2

3

4

5

6

28.2: Nucleosides. N-Glycosides of a purine or pyrimidine heterocyclic base and a carbohydrate. The C-N bond involvesthe anomeric carbon of the carbohydrate. The carbohydrates for nucleic acids are D-ribose and 2-deoxy-D-ribose

343

Nucleosides = carbohydrate + base (Table 28.2, p. 1168) ribonucleosides or 2’-deoxyribonucleosides

OHO N

HO

N

N

N

X

NH2

OHO N

HO

N

N

NH

X

O

NH2

RNA: X= OH, adenosine (A)DNA: X= H, 2'-deoxyadenosine (dA)

RNA: X= OH, guanosine (G)DNA: X= H, 2'-deoxyguanosine (dG)

OHO

HO X

N

N

O

NH2

OHO

HO

N

NH

O

O

H3C

RNA: X= OH, cytidine (C)DNA: X= H, 2'-deoxycytidine (dC)

DNA: thymidine (T)

OHO

HO

N

NH

O

O

RNA: R= H, uridine (U)

1

2

3

4

567

89

1'

2'3'

4'

5'

OH

1'

2'3'

4'

5' 2

1

3

4

5

6

To differentiate the atoms of the carbohydrate from the base, theposition number of the carbohydrate is followed by a ´ (prime).

The stereochemistry of the glycosidic bond found in nucleic acids is β.

176

344

28.3: Nucleotides. Phosphoric acid esters of nucleosides.Nucleotides = nucleoside + phosphate

O

HO X

HO B

ribonucleoside (X=OH)deoxyribonucleoside (X=H)

ribonucleotide 5'-monophosphate (X=OH, NMP)deoxyribonucleotide 5'-monophosphate (X=H, dNMP)

O

HO X

O BPHO

O

O

O

HO X

O BPO

O

O

PHO

O

O

O

HO X

O BPO

O

O

PO

O

O

PHO

O

O

ribonucleotide 5'-diphosphate (X=OH, NDP)deoxyribonucleotide 5'-diphosphate (X=H, dNDP)

ribonucleotide 5'-triphosphate (NTP)deoxyribonucleotide 5'-triphosphate (X=H, dNTP)

O

O OH

O B

P

O

O

ribonucleotide3',5'-cyclic phosphosphate (cNMP)

Kinase: enzymes that catalyze the phosphoryl transfer reactionfrom ATP to an acceptor substrate. M2+ dependent

OHOHO

OHOH

OH

O N

N

N

N

NH2

HO OH

OPO

O

O

P

O

O

O

PO

O

O

OHOHO

OHOH

OPO32-

ATP

O N

N

N

N

NH2

HO OH

OPO

O

O

P

O

O

O

ADPGlucose-6-phosphate

Glucose

345

O N

N

N

N

NH2

HO OH

OPO

O

O

P

O

O

O

PO

O

O

ATP

O N

N

N

N

NH2

HO OH

OPO

O

O

AMP

O

O OH

O

P

O

O

N

N

N

N

NH2

-P2O7

adenylylcyclase H2O

cAMP

O N

N

N

NH

O

HO OH

OPO

O

O

P

O

O

O

PO

O

O

GTP

O N

N

N

NH

O

HO OH

OPO

O

O

GMP

O

O OH

O

P

O

O

N

N

N

NH

O

-P2O7

guanylatecyclase H2O

cGMP

NH2 NH2 NH2

1971 Nobel Prize in Medicine or Physiology: Earl Sutherland

28.4: Bioenergetics. (Please read)28.5: ATP and Bioenergetics. (Please read)

+ HPO4 2-O N

N

N

N

NH2

HO OH

OPO

O

O

P

O

O

O

PO

O

O

ATP

H2O O N

N

N

N

NH2

HO OH

OPO

O

O

P

O

O

O

ADP

+ ~31 KJ/mol (7.4 Kcal/mol)

177

346

28.6: Phosphodiesters, Oligonucleotides, and Polynucleotides. The chemical linkage betweennucleotide units in nucleic acids is a phosphodiester,which connects the 5’-hydroxyl group of one nucleotide to the 3’-hydroxyl group of the next nucleotide.

By convention, nucleic acid sequences are writtenfrom left to right, from the 5’-end to the 3’-end.

Nucleic acids are negatively charged

28.7: Nucleic Acids. 1944: Avery, MacLeod & McCarty - Strong evidence that DNA

is genetic material1950: Chargaff - careful analysis of DNA from a wide variety of

organisms. Content of A,T, C & G varied widely accordingto the organism, however: A=T and C=G (Chargaff’ Rule)

1953: Watson & Crick - structure of DNA (1962 Nobel Prize with M. Wilkens, 1962)

RNA, X= OHDNA, X=H

O

O

O X

Base

PO

O

O

O

O X

Base

P

O

O

O

3'

3'

5'

5'

3'

5'

347

28.8: Secondary Structure of DNA: The Double Helix.Initial “like-with-like”, parallel helix: Does not fit with with Chargaff’s Rule: A = T G = C

Wrong tautomers !!

Watson, J. D. The Double Helix, 1968

N

N

N

N

N

H

dR

N

N

N

N

N

H

dR

H

N

N

dR

OH

O

N

N

dR

OH

O

purine - purine pyrimidine - pyrimidine

N

N

dR

NH

O

N

N

dR

NH

O

H

N

N

N

N

O

N

H

dR

N

N

N

N

O

H

dR

H

H

H

N

H

H

H

178

348

Two polynucleotide strands, running in opposite directions (anti-parallel) and coiled around each other in a double helix.

The strands are held together by complementary hydrogen- bonding between specific pairs of bases.

O

OR

RON

N

N

O

H

H

O

OR

RO

N

NN

NO

N

H

H

H

Hydrogen BondDonor

Hydrogen BondDonor

Hydrogen BondAcceptor

Hydrogen BondAcceptor

3'

5'

Hydrogen BondAcceptor

Hydrogen BondDonor

5'

O2

N4 O6

3'

Antiparallel C-G Pair

N3 N1

N2

O

OR

RON

N

O

O

O

OR

RO

N

N N

NN

H

H

H

5'

3'

3'

5'

Hydrogen BondDonor

N6

Hydrogen BondAcceptor

Hydrogen BondAcceptor O4

Hydrogen BondDonor

Antiparallel T-A Pair

N1N3

349

DNA double helix

one helical

turn34 Å

major groove12 Å

minor groove

6 Å

backbone: deoxyribose and phosphodiester linkagebases

179

350

28.9: Tertiary Structure of DNA: Supercoils. Each cell contains about two meters of DNA. DNA is “packaged” by coiling around a core of proteins known as histones. The DNA-histone assembly is called a nucleosome. Histones are rich is lysine and arginine residues.

Pdb code 1kx5

351

28.10: Replication of DNA.The Central Dogma (F. Crick):

DNA replication DNA transcription mRNA translation Protein (genome) (transcriptome) (proteome)

Expression and transfer of genetic information:Replication: process by which DNA is copied with very high

fidelity. Transcription: process by which the DNA genetic code is read

and transferred to messenger RNA (mRNA). This is an intermediate step in protein expression

Translation: The process by which the genetic code is converted to a protein, the end product of gene expression.

The DNA sequence codes for the mRNA sequence, whichcodes for the protein sequence

“It has not escaped our attention that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson & Crick

180

352

DNA is replicated by the coordinated efforts of a number of proteins and enzymes.

For replication, DNA must be unknotted, uncoiled and thedouble helix unwound.

Topoisomerase: Enzyme that unknots and uncoils DNAHelicase: Protein that unwinds the DNA double helix. DNA polymerase: Enzyme that replicates DNA using each strand

as a template for the newly synthesized strand.DNA ligase: enzyme that catalyzes the formation of the

phosphodiester bond between pieces of DNA.

DNA replication is semi-conservative: Each new strand of DNAcontains one parental (old, template) strand and onedaughter (newly synthesized) strand

353

Unwinding of DNA by helicases expose the DNA bases (replication fork) so that replication can take place. Helicasehydrolyzes ATP in order to break the hydrogen bonds betweenDNA strands

DNA replication

181

354

DNA Polymerase: the new strand is replicated from the 5’ → 3’(start from the 3’-end of the template)

DNA polymerases are Mg2+ ion dependent

The deoxynucleotide 5’-triphosphate (dNTP) is the reagent for nucleotide incorporation

G

O

O

OP

O

-O

A

O

O

O

T

O

OH

O P O

O

C

O

OH

O

O-PO-

O

O P

O-

O

O-

Mg2+

5'

3'

5'

templatestrand(old)

(new)

3’-hydroxyl group of the growing DNA strand acts as a nucleophile and attacks the α-phosphorus atom of the dNTP.

dNTP

355

Replication of the leading strand occurs continuously in the 5’ → 3’ direction of the new strand.

Replication of the lagging strand occurs discontinuously. ShortDNA fragments are initially synthesized and then ligated together. DNA ligase catalyzes the formation of the phosphodiester bond between pieces of DNA.

animations of DNA processing: http://www.wehi.edu.au/education/wehi-tv/dna/

182

356

DNA replication occurs with very high fidelity:Most DNA polymerases have high intrinsic fidelityMany DNA polymerases have “proof-reading”

(exonuclease) activityMismatch repair proteins seek out and repair base-pair

mismatches due to unfaithful replication

28.11 Ribonucleic AcidRNA contains ribose rather than 2-deoxyribose and uracil rather than thymine. RNA usually exist as a single strand.

There are three major kinds of RNAmessenger RNA (mRNA): ribosomal RNA (rRNA)transfer RNA (tRNA)

DNA is found in the cell nucleus and mitochondria; RNA is moredisperse in the cell.

357

Transcription: only one of the DNA strands is copied (codingor antisense strand). An RNA polymerase replicates the DNA sequence into a complementary sequence of mRNA (template or sense strand). mRNAs are transported from the nucleus to the cytoplasm, where they acts as the template for protein biosynthesis (translation). A three base segment of mRNA (codon) codes for an amino acid.The reading frame of the codons is defined by the start and stop codons.

183

358

5'-cap 5'-UTR start stop 3'-UTRCoding sequence

The mRNA is positioned in the ribosome through complementarypairing of the 5’-untranslated region of mRNA with a rRNA.

Transfer RNA (tRNA): t-RNAs carries an amino acid on the 3’-terminal hydroxyl (A) (aminoacyl t-RNA) and the ribosome catalyzes amide bond formation.

Ribosome: large assembly of proteins and rRNAs that catalyzesprotein and peptide biosynthesis using specific, complementary,anti-parallel pairing interactions between mRNA and the anti-codon loop of specific tRNA’s.

359

Although single-stranded, there are complementary sequences within tRNA that give it a defined conformation.

The three base codon sequence of mRNA are complementary to the “anti-codon” loops of the appropriate tRNA. The base-pairing between the mRNA and the tRNA positions the tRNAs for amino acid transfer to the growing peptide chain.

aminoacyl t-RNA

TψC loop

D loop

variable loop

184

360

28.12: Protein Biosynthesis. Ribosomal protein synthesis

P siteU G U AA U C U CG U U

3'5'

A siteA CU

OO

HN

SCH3

OHC

U G U AA U C U CG U U

3'5'

A CU

OO

HN

SCH3

OHC

U AA

OO

H2N

OH

U G U AA U C U CG U U

3'5'

U AA

OO

HN

OHONH

CH3S

A CU

OH

OHC

U G U AA U C U CG U U

3'5'

U AA

OO

HN

OHONH

CH3S

A CU

OH

OHC

U G U AA U C U CG U U

3'5'

U AA

OO

HN

OHONH

CH3S

A CU

OH

OHC

E siteE site

U G U AA U C U CG U U

3'5'

U AA

OO

HN

OHONH

CH3S

OHC

G AC

O O

CH3H2N

U G U AA U C U CG U U

3'5'

O

HN

OH

O

HN

G AC

O O

CH3HN

SCH3

U AA

OH

OJHC

U G U AA U C U CG U U

3'5'

O

HN

OH

O

HN

G AC

O O

CH3HN

SCH3

U AA

OH

OHC

U G U AA U C U CG U U

3'5'

O

HN

OH

O

HN

G AC

O O

CH3HN

SCH3

U AA

OH

OHC

361

28.13: AIDS. (please read)28.14: DNA Sequencing. Maxam-Gilbert: relys on reagents that react with a specific DNA

base that can subsequent give rise to a sequence specific cleavage of DNA

Sanger: Enzymatic replication of the DNA fragment to be sequenced with a DNA polymerase, Mg+2, and dideoxynucleotides triphosphate (ddNTP) that truncates DNA replication

Restriction endonucleases: Bacterial enzymes that cleaveDNA at specific sequences

5’-d(G-A-A-T-T-C)-3’3’-d(C-T-T-A-A-G)-5’

5’-d(G-G-A-T-C-C)-3’3’-d(C-C-T-A-G-G)-5’

EcoR I

BAM HI

5'

3'

3'

5'

5'

3'

3'

5'

OH 2-O3PO

2-O3PO OH

restrictionenzyme

185

362

Sanger Sequencing:key reagent: dideoxynucleotides triphosphates (ddNTP)

N

NN

N

NH2

OOPO

O-

O

P

O

O-

OP

O

-O

O-

NH

NN

N

O

OOPO

O-

O

P

O

O-

OP

O

-O

O-

ddATP

NH2

ddGTP

OOPO

O-

O

P

O

O-

OP

O

-O

O-

OOPO

O-

O

P

O

O-

OP

O

-O

O-

ddTTP ddCTP

N

NH

O

ON

N

NH2

O

When a ddNTPis incorporatedelongation of the primer is terminated

The ddNTP isspecificallyincorporatedopposite itscomplementarynucleotide base

DNA polymerase

3'

5'-32P3'

5'

primer

Template

unknown sequence

Mg2+, dNTP's

+ one ddATP

O

N3

HO N

NH

O

OOHO N

N

N

NH

O

S

O OHN

N

H2N

OOHO N

N

NH2

O

ddIAZT

(-)-3TC

Anti-Viral Nucleosides

d4C

363

Sanger Sequencing

Larger fragments

Smallerfragments

G

T

A

A

C

G

T

A

A

T

C

A

C

A

G

ddA ddG ddC ddT

C

A

T

T

G

C

A

T

T

A

G

T

G

T

C

5'

32P

3'

5'

3'

32P-5' 3'

primer template

186

364

28.15: The Human Genome Project. (please read)28.16: DNA Profiling and Polymerase Chain Reaction (PCR).method for amplifying DNA using a DNA polymerase, dNTPs and cycling the temperature.

Heat stable DNA Polymerases (from archaea):Taq: thermophilic bacteria (hot springs)- no proof readingPfu: geothermic vent bacteria- proof reading

Mg 2+

two Primer DNA strands (synthetic, large excess)one sense primer and one antisense primer

one Template DNA strand (double strand)dNTP’s

1 x 2 = 2 x 2 = 4 x 2 = 8 x 2 = 16 x 2 = 32 x 2 = 64 x 2 = 128 x 2 = 256 x 2 = 512 x 2 = 1,024 x 2 = 2,048 x 2 = 4,096 x 2 = 8,192 x 2 = 16,384 x 2 = 32,768 x 2 = 65,536 x 2 = 131,072 x 2 = 262,144 x 2 = 524,288 x 2 = 1,048,576

In principle, over one million copies per original, can be obtained after just twenty cycles

KARY B. MULLIS, 1993 Nobel Prize in Chemistry for his invention of the polymerase chain reaction (PCR) method.

365

Polymerase Chain Reaction

For a PCR animation go to: http://www.blc.arizona.edu/INTERACTIVE/recombinant3.dna/pcr.html http://users.ugent.be/~avierstr/principles/pcrani.html

5'

3'

3'

5'

95 °C

denaturation

5' 3'

3' 5'

anneal (+) and (-) primers

55 - 68 °C

5' 3'

3' 5'

3'

3'

72 °C

Taq, Mg 2+, dNTPs

extension

5'

3'

3'

5'

5'

3'

3'

5'

95 °C

denaturation

2nd cycle

5'

3'

3'

5'

5'

3'

3'

5'

amplification of DNA

2 copies of DNA

repeat temperature cycles