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Home , Deoxyribonucleoside, Deoxyribonucleotide, Deoxyribose, Nucleic acid, Nucleoside, Nucleotide

Chapter 28: , , and Nucleic . Nucleic acids are the third class of ( and being the others) Two major classes of nucleic acids deoxyribonucleic (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 () () (proteome)

The monomeric units for nucleic acids are nucleotides Nucleotides are made up of three structural subunits 1. : in RNA, 2- in DNA 2. Heterocyclic base 3.

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Nucleoside, nucleotides and nucleic acids

phosphate

sugar base

phosphate phosphate

sugar base sugar base sugar base

phosphate nucleotides

sugar base

nucleic acids

The chemical linkage between units in nucleic acids is a phosphodiester 341

174 28.1: and . 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 )

NH2 O 7 6 N 5 1 N N N 8 N NH 2 9 N 4 N N N N N NH H 3 H H 2 (A) (G) DNA/RNA DNA/RNA

NH2 O O 4 H3C 5 N 3 N NH NH

6 2 N N O N O N O 1 H H H (C) (T) (U) DNA/RNA DNA RNA 28.2: Nucleosides. N-Glycosides of a purine or pyrimidine heterocyclic base and a . The C-N bond involves the anomeric of the carbohydrate. The for nucleic acids are D-ribose and 2-deoxy-D-ribose 342

Nucleosides = carbohydrate + base (Table 28.2, p. 1168) or 2’- NH2 O 7 6 N 5 1 N N NH 8 5' 9 HO N 2 O 4 N HO O N N NH 3 2 4' 1' 3' 2' HO X HO X RNA: X= OH, (A) RNA: X= OH, (G) DNA: X= H, 2'- (dA) DNA: X= H, 2'- (dG)

NH2 O O 4 H3C 5 N 3 NH NH 6 1 HO 5' N 2 O N O HO N O O HO O O 4' 1' 3' 2' HO X HO HO OH RNA: X= OH, (C) DNA: (T) RNA: R= H, (U) DNA: X= H, 2'- (dC) To differentiate the of the carbohydrate from the base, the position number of the carbohydrate is followed by a ´ (). The of the found in nucleic acids is β. 343

175 28.3: Nucleotides. Phosphoric acid esters of nucleosides. Nucleotides = nucleoside + phosphate O HO O B HO P O O B O

HO X HO X

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

O O O O O HO P O P O P O HO P O P O O B O B O O B O O O O O O P HO X HO X O O OH ribonucleotide 5'-diphosphate (X=OH, NDP) ribonucleotide 5'-triphosphate (NTP) ribonucleotide deoxyribonucleotide 5'-diphosphate (X=H, dNDP) deoxyribonucleotide 5'-triphosphate (X=H, dNTP) 3',5'-cyclic phosphosphate (cNMP)

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

NH OH O O O N 2 OPO 2- 3 O O N NH2 HO O P O P O P O O N HO O N O O P O P O HO O O O HO O N N N O O OH OH N OH HO OH OH HO OH ATP Glucose-6-phosphate ADP

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NH O O O N NH2 adenylyl N NH2 O N 2 H2O O P O P O P O cyclase O P O O N N O O N N O N N O O O O N O N N -P2O7 P HO OH O O OH HO OH ATP cAMP AMP O O O O N O guanylate N O O N H2O O P O P O P O cyclase O P O O N NH O O N NH O N NH O O O O N O N N -P2O7 P NH NH NH2 HO OH 2 O O OH 2 HO OH GTP cGMP GMP

1971 Nobel Prize in Medicine or Physiology: Earl Sutherland 28.4: Bioenergetics. (Please read) 28.5: ATP and Bioenergetics. (Please read)

NH O O O N NH2 O O N 2 O P O P O P O H O O P O P O O N N 2 O N N + HPO 2- + ~31 KJ/mol O O 4 O O O N N (7.4 Kcal/mol) HO OH HO OH ATP ADP

345

176 28.6: Phosphodiesters, , and 3' O . The chemical linkage between O P O O Base units in nucleic acids is a phosphodiester, 5' O which connects the 5’-hydroxyl group of one 3' O X nucleotide to the 3’-hydroxyl group of O P O O Base the next nucleotide. 5' O

3'

By convention, sequences are written O X from left to right, from the 5’-end to the 3’-end. 5' Nucleic acids are negatively charged RNA, X= OH DNA, X=H 28.7: Nucleic Acids. 1944: Avery, MacLeod & McCarty - Strong evidence that DNA is genetic material 1950: Chargaff - careful analysis of DNA from a wide variety of . Content of A,T, C & G varied widely according to the , however: A=T and C=G (Chargaff’ Rule) 1953: Watson & Crick - structure of DNA (1962 Nobel Prize with M. Wilkens, 1962) 346

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

H H N N N dR dR dR dR N O N N N N O N H N O N N N N H H H O O H H H N N H H H H N N N O N N H N O N N O N N N dR dR dR dR N N N H H purine - purine pyrimidine - pyrimidine

Wrong !!

Watson, J. D. The Double Helix, 1968 347

177 Two strands, running in opposite directions (anti-parallel) and coiled around each other in a double helix. The strands are held together by complementary - bonding between specific pairs of bases.

Antiparallel C-G Pair

Hydrogen Bond 4 H 6 Donor N O N H O N Acceptor Hydrogen Bond 5' N3 N1 Hydrogen Bond Acceptor N H N N RO Donor 5' N N O Hydrogen Bond 2 RO O Hydrogen Bond O O H N 2 Acceptor N Donor H OR 3' OR 3'

Antiparallel T-A Pair H Hydrogen Bond 6 Hydrogen Bond 4 O H N N Acceptor O N Donor 5' Hydrogen Bond N3 N H N N RO N1 Hydrogen Bond Donor 5' Acceptor N N O RO O O OR 3' OR 348 3'

DNA double helix

major groove 12 Å one helical turn 34 Å minor groove 6 Å

backbone: deoxyribose and phosphodiester linkage bases 349

178 28.9: Tertiary Structure of DNA: Supercoils. Each contains about two meters of DNA. DNA is “packaged” by coiling around a core of proteins known as . The DNA- assembly is called a nucleosome. Histones are rich is lysine and arginine residues.

350 Pdb 1kx5

“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 28.10: Replication of DNA. The Central Dogma (F. Crick): DNA replication DNA mRNA 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 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 expression. The DNA sequence for the mRNA sequence, which

codes for the protein sequence 351

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

For replication, DNA must be unknotted, uncoiled and the double helix unwound. Topoisomerase: that unknots and uncoils DNA : Protein that unwinds the DNA double helix. DNA : Enzyme that replicates DNA using each strand as a template for the newly synthesized strand. DNA ligase: enzyme that catalyzes the formation of the 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

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Unwinding of DNA by 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

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180 DNA Polymerase: the new strand is replicated from the 5’ → 3’ (start from the 3’-end of the template) DNA are Mg2+ ion dependent The deoxynucleotide 5’-triphosphate (dNTP) is the reagent for nucleotide incorporation

OH dNTP 5' O O O O O P O P O P O- - A T O O- O- O O OH Mg2+

O O O O P G C -O O (new) O 5' template strand O (old) 3' 3’-hydroxyl group of the growing DNA strand acts as a nucleophile and attacks the α- of the dNTP. 354

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.

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

181 DNA replication occurs with very high fidelity: Most DNA polymerases have high intrinsic fidelity Many DNA polymerases have “proof-reading” () activity Mismatch repair proteins seek out and repair base-pair mismatches due to unfaithful replication 28.11 Ribonucleic Acid RNA contains ribose rather than 2-deoxyribose and uracil rather than thymine. RNA usually exist as a single strand.

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

DNA is found in the and mitochondria; RNA is more disperse in the cell. 356

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 , where they acts as the template for protein (translation). A three base segment of mRNA (codon) codes for an .The of the codons is defined by the start and stop codons.

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182 5'-cap 5'-UTR start Coding sequence stop 3'-UTR

The mRNA is positioned in the through complementary pairing of the 5’-untranslated region of mRNA with a rRNA. Transfer RNA (tRNA): t- carries an amino acid on the 3’-terminal hydroxyl (A) (aminoacyl t-RNA) and the ribosome catalyzes bond formation.

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

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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.

variable loop

TψC loop

D loop

aminoacyl t-RNA 359

183 28.12: . Ribosomal protein synthesis

SCH3 SCH3 OH H N H OHC N H N OHC 2 O O O O O O

A site P site U A C U A C A U A A U G U A U G C U C U U A U G U A U G C U C U U 5' 3' 5' 3'

SCH3

OOJHC HN CH S 3 O CH3S HN OH OH OHC O OH OHC O OH N O N H H N CH OH H HN H2N CH3 HN

O OH O O OH O O

A U A

U A C

C A A U A C G A U C U G A A U A A U G U A U G C U C U U A U G U A U G C U C U U 55''' 3'' 5'' 3'' E site 360

28.13: AIDS. (please read) 28.14: DNA . 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 cleave DNA at specific sequences

5' 3'

3' 5' 5’-d(G-A-A-T-T-C)-3’ 3’-d(C-T-T-A-A-G)-5’ EcoR I restriction enzyme

2- OH O3PO 5’-d(G-G-A-T-C-C)-3’ 5' 3' 3’-d(C-C-T-A-G-G)-5’ BAM HI 3' 5' 2- O3PO OH 361

184 : key reagent: dideoxynucleotides triphosphates (ddNTP)

O NH2 NH2 O N N N N NH NH O O O N O O O N O O O O N O O O N NH2 N N O - - - - O P O P O P O O P O P O P O O P O P O P O O O P O P O P O O O O ------O- O- O- O- O- O- O O O O O O

ddATP ddTTP ddGTP ddCTP

32 3' 5'- P Anti-Viral Nucleosides primer When a ddNTP O O is incorporated N 3' NH NH elongation of HO N O HO O N the primer is O N terminated N3 DNA polymerase AZT ddI The ddNTP is Mg2+, dNTP's NH2 H2N + one ddATP specifically incorporated N N HO N O N O OH opposite its O O complementary S nucleotide base d4C (-)-3TC

5' Template unknown sequence 362

Sanger Sequencing ddA ddG ddC ddT 5' Larger fragments C G A T T A T A G C C G A T T A T A A T G C T A G C Smaller T A fragments C G 3'

5' 3' 32P 32P-5' 3' primer template

363

185 28.15: The Human . (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 ): Taq: thermophilic (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 364 polymerase chain reaction (PCR) method.

Polymerase Chain Reaction 5' 3'

5' 3' 95 °C

denaturation 3' 5'

3' 5'

5' 3' 72 °C 55 - 68 °C Taq, Mg 2+, dNTPs 3' anneal 3' extension (+) and (-) primers 3' 5'

5' 3'

5' 3'

3' 5' 95 °C 3' 5' 5' 3' denaturation 5' 3' 3' 5' 2nd cycle

2 copies of DNA 3' 5' repeat temperature cycles

amplification of DNA

For a PCR animation go to: http://www.blc.arizona.edu/INTERACTIVE/recombinant3.dna/pcr.html http://users.ugent.be/~avierstr/principles/pcrani.html 365

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