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25.19: Coenzymes. Some reactions require additional organic molecules or metal ions. These are referred to as cofactors or coenzymes.

N N

S OP

HO O

O P OH

OH

O

NH2N

+

Thiamin Diphosphate(vitamin B1)

OPOHO

HON

O

OH

H

Pyridoxal Phophates(vitamin B6)

N

NN

N

OHO O

OH

Fe

Heme

S

NHHN

O

CO2H

Biotin(vitamin B7)

NN

N N

H2N

O NH2

O

NH2

NH2OO

OCo

O

H

H2N

O

NH

O

C

P OO-O O

N

N

HO H

H

NH2

H

H

OH

N

Vitamin B12(cyanocobalamin)

HN

N

N

N

HN

H2N

OHN

O CO2-

CO2-

Folic Acid(vitamin B9)

N

N

NH

N O

O

O

OH

HOOH

O-

P

O

P OO

O-O

HO

N

OH

O

N

N

N

NH2

Flavin Adenine Diphosphate (FAD)(Vitamin B2)

25.20: Protein Quaternary Structure: Hemoglobin. (please read) 25.21: G-Coupled Protein Receptors. (please read)

358

Chapter 26: 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 information.

ribonucleic acid (RNA): an intermediate in the expression of genetic information and other diverse roles.

The Central Dogma (F. Crick):

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

The monomeric units for nucleic acids are nucleotides. Nucleotides are made up of three structural subunits

1. Sugar: ribose in RNA, 2-deoxyribose in DNA 2. Heterocyclic base 3. Phosphodiester

transcription translation

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359

Nucleoside, nucleotides and nucleic acids

sugar base

phosphodiester

sugar base

sugar base

phosphodiester

phosphodiester

sugar base

phosphate

sugar base

Nucleoside Nucleotide

Nucleic Acid

360

26.1: Pyrimidines and Purines. The heterocyclic bases; there are five six common bases for nucleic acids (Table 26.1, p. 1087). Note that G, T and U exist in the keto form (and not the enol form found in phenols)

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

N

NH N

N

N

N

purine

pyrimidine

N

NH N

N N

NH N

NH

NH2 O

NH2

NH

N

O

NH2

NH

NH

O

O

NH

NH

O

OH3C

adenine (A)DNA/RNA

guanine (G)DNA/RNA

cytosine (C)DNA/RNA

thymine (T)DNA

uracil (U)RNA

1

2

34

567

8

9

12

34

5

6 NH

N

O

NH2

5-methylcytosine (C)DNA

H3C

167

361

Nucleosides = carbohydrate + base (Table 28.2, p. 1089) ribonucleosides or 2’-deoxyribonucleosides

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

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

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

OH3C

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

DNA: thymidine (T)

OHO

HO

N

NH

O

O

RNA: uridine (U)

1

2

34

567

8 9

1'2'3'

4'

5'

OH

1'2'3'

4'

5' 21

34

5

6

OHO

HO

N

N

O

NH2H3C

DNA: 5-methylcytosineDNA: 5-methyl-2’-deoxycytidine

362

26.3: 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 BPHOO

O

O

HO X

O BPOO

OPHOO

OO

HO X

O BPOO

OPOO

OPHOO

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

PO

O

ribonucleotide3',5'-cyclic phosphosphate (cNMP)

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

OHOHO

OHOH

OHO N

N

NN

NH2

HO OH

OPOO

OPO

OO

POO

O

OHOHO

OHOH

OPO32-

ATP

O N

N

NN

NH2

HO OH

OPOO

OPO

OO

ADPGlucose-6-phosphateGlucose

hexokinase -or-

glucokinase

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363

O N

N

NN

NH2

HO OH

OPOO

OPO

OO

POO

O

ATP

O N

N

NN

NH2

HO OH

OPOO

O

AMP

O

O OH

O

PO

ON

N

NN

NH2

-P2O7

adenylylcyclase H2O

cAMP

O N

N

NNH

O

HO OH

OPOO

OPO

OO

POO

O

GTP

O N

N

NNH

O

HO OH

OPOO

O

GMP

O

O OH

O

PO

ON

N

NNH

O

-P2O7

guanylatecyclase H2O

cGMP

NH2 NH2 NH2

1971 Nobel Prize in Medicine or Physiology: Earl Sutherland

26.4: Bioenergetics. (Please read)

26.5: ATP and Bioenergetics. (Please read)

+ HPO4 2-O N

N

NN

NH2

HO OH

OPOO

OPO

OO

POO

O

ATP

H2O O N

N

NN

NH2

HO OH

OPOO

OPO

OO

ADP

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

364

26.6: Phosphodiesters, Oligonucleotides, and Polynucleotides. The chemical linkage between nucleotide units of 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 written from left to right, from the 5’-end to the 3’-end.

Nucleic acids are negatively charged

RNA, X= OH DNA, X=H

5’-direction

3’-­‐direc)on  

OO

O X

N

POO

OO

HO X

N

PO

OO

3'

3'

5'

5'

3'

5'

OH

-H2O

OO

O X

N

POO

OO

O X

PO

OO

3'

3'

5'

5'

3'

5'

N

NH2

O

N

O

NH2

N

N

N

NH2

N

N

N

N

NH2

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365

26.7: Nucleic Acids (please read). Chargaff’s Rule: A=T and C=G

26.8: Secondary Structure of DNA: The Double Helix. 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

HH

O

OR

RO

NN N

NO

NH

H

HHydrogen Bond

Donor

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

RO NN

O

OO

OR

RON

N N

NN

H

HH

5'

3'

3'

5'

Hydrogen BondDonorN6

Hydrogen BondAcceptor

Hydrogen BondAcceptor O4

Hydrogen BondDonor

Antiparallel T-A Pair

N1N3

(Fig. 26.3, p. 1097)

366

DNA double helix

one helical turn 34 Å

major groove 12 Å

minor groove

6 Å

backbone: deoxyribose and phosphodiester linkage bases

(Figs. 26.4 & 26.5, p. 1098)

170

367

26.9: 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

368

26.10: 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, which codes 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

171

369

DNA is replicated by the coordinated efforts of multiple proteins and enzymes. For replication, DNA must be unknotted, uncoiled and the double helix unwound. Topoisomerase: Enzyme that unknots and uncoils DNA

Helicase: 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 DNA contains one parental (old, template) strand and one daughter (newly synthesized) strand

370

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

DNA replication

(Fig. 26.8, p. 1100)

http://www.hhmi.org/biointeractive/dna-replication-advanced-detail

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371

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

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

dNTP

(Fig 26.9, p. 1101)

G

O

O

OPO

-O

A

O

O

OT

O

OH

O P OO

C

O

OH

O

O-PO-

OO PO-

OO-

Mg2+

5'

3'

5'templatestrand(old)

(new)

372

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

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

(Fig. 26.8, p. 1100)

173

373

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

There are three four major kinds of RNA: messenger RNA (mRNA): ribosomal RNA (rRNA) transfer RNA (tRNA) microRNA (miRNA)

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

374

Transcription: only one of the DNA strands is copied (coding or 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.

(Table 26.4, p. 1103)

174

375

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

The mRNA is positioned in the ribosome through complementary pairing 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 catalyzes protein and peptide biosynthesis using specific, complementary, anti-parallel pairing interactions between mRNA and the anti-codon loop of specific tRNA’s.

376

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

(Fig. 26.11, p. 1104)

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377

26.12: Protein Biosynthesis. Ribosomal protein synthesis

P site U G U AA U C U CG U U

3'5'

A site A 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 U3'5'

U AA

OO

HN

OHONH

CH3S

A CU

OH

OHC

U G U AA U C U CG U U3'5'

U AA

OO

HN

OHONH

CH3S

A CU

OH

OHC

U G U AA U C U CG U U3'5'

U AA

OO

HN

OHONH

CH3S

A CU

OHOHC

E site U G U AA U C U CG U U3'5'

U AA

OO

HN

OHONH

CH3S

OHC

G AC

O O

CH3H2N

U G U AA U C U CG U U3'5'

O

HNOH

OHN

G AC

O O

CH3HN

SCH3

U AA

OH

OJHC

U G U AA U C U CG U U3'5'

O

HNOH

OHN

G AC

O O

CH3HN

SCH3

U AA

OH

OHC

U G U AA U C U CG U U3'5'

O

HNOH

OHN

G AC

O O

CH3HN

SCH3

U AA

OH

OHC

(Fig. 26.12, p. 1105)

https://www.hhmi.org/biointeractive/translation-advanced-detail

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26.13: AIDS. (please read) 26.14: DNA Sequencing. Maxam-Gilbert: relies 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 cleave DNA 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

176

379

Sanger Sequencing: key reagent: dideoxynucleotides triphosphates (ddNTP)

N

NN

N

NH2

OOPOO-

OPO

O-OP

O-O

O-

NH

NN

N

O

OOPOO-

OPO

O-OP

O-O

O-

ddATP

NH2

ddGTP

OOPOO-

OPO

O-OP

O-O

O-OOPO

O-

OPO

O-OP

O-O

O-

ddTTP ddCTP

N

NH

O

ON

N

NH2

O

When a ddNTP is incorporated elongation of the primer is terminated The ddNTP is specifically incorporated opposite its complementary nucleotide base

DNA polymerase

3'

5'-32P3'

5'

primer

Templateunknown sequence

Mg2+, dNTP's+ one ddATP

O

N3

HO NNH

O

OOHO N

N

N

NH

O

S

O OHNN

H2N

OOHO N

N

NH2

O

ddIAZT

(-)-3TC

Anti-Viral Nucleosides

d4C

380

Sanger Sequencing

Larger fragments

Smaller fragments

GTAACGTAATCACAG

ddA ddG ddC ddT

CATTGCATTAGTGTC

5'32P

3'

5'

3'

32P-5' 3'

primer template

http://www.hhmi.org/biointeractive/sanger-method-dna-sequencing

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381

26.15: The Human Genome Project. (please read) 26.16: 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 reading Pfu: 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.

382

Polymerase Chain Reaction

For a PCR animation go to: http://www.youtube.com/watch?v=ZmqqRPISg0g

5'

3'

3'

5'

95 °C

denaturation

5' 3'

3' 5'

anneal (+) and (-) primers

55 - 68 °C5' 3'

3' 5'

3'3'

72 °CTaq, 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

(Fig. 26.14, p. 1109-10)

178