Structure of Nucleic Acids, and Functional Significance

EDWARD WAMPANDE PhD Course Outline NUCLEIC ACIDS 1. Structure and function of DNA

2. Structure and function of .

3. Basic structure: Sugars, Bases, phosphate

4. Biomedical importance of Synthetic analogues

5. Biological importance of nucleic acids

Nucleic Acids

• Nucleic acids – DNA and RNA – Polymers of

– NB: other biomolecules that are polymers of simple units; , CHOs, Lipids

NB; Nucleic in plant, invertebrates vertebrates is the same History of DNA • DNA was first identified and isolated by Friedrich Miescher in 1869 at the University of Tübingen, a substance he called ‘nuclein’

• In 1919, Phoebus Levene identified the base, sugar and phosphate unit.

• In 1937, produced the first X- ray diffraction patterns that showed that DNA had a regular structure.

• Double helix structure of DNA was first discovered in 1953 by Watson and Crick at the University of Cambridge, using experimental data collected by and Maurice ... • Sense strand and antisense strand-Polarity & direction

Nucleic Acids structure • Primary structure –the components of Nucleic Acids – The 5-Carbon sugars – Nitrogenous Bases – The Phosphate Functional group

• Secondary structure: the double helix

• Tertiary structure: Supercoiling Primary structure

• The 5-Carbon sugars

• Nitrogenous Bases

• The Phosphate Functional group

• Nucleotides and Nucleosides 5-C Sugars in Nucleic Acids

• The 5-C atoms numbered 1’ to 5’; primes are used to distinguish with numbering in nitrogenous bases • Both sugars have an O2; 5’-C is outside the ring

• Sugars differ in Presence or Absence of an O2 in 2’-C The Nitrogenous bases

Bases: N2-containing molecules having the chemical properties of a base a substance that accepts an H+ ion or proton in solution)

Tautomeric forms of uracil. The lactam form predominates at pH 7.0; the other forms become more prominent as pH decreases.

The Phosphate functional group

It gives DNA and RNA the property of an acid (a substance that releases a H+ in solution), hence the name nucleic acids Complimentary rule

• -

• - Watson-Crick base-pairing in DNA

2 bonds

3 bonds Significance of complementary base pairing

• Ensures storage, preservation and transmission of genetic information. The arrangement of bases determine: – a) functionality of DNA; i.e., vs. non-genes; – b) organismal/species identity; – c) evolution

• Each DNA strand is the predictable counterpart of the other – Once you know the sequence of one strand, you can easily figure out the sequence of the other strand

• DNA replication; each of the two daughter DNA molecules have one old strand derived from the parent and one newly made strand – Complementary base pairing results in the two daughter DNA molecules being identical.

“Chargaff’s rules 1.The base composition of DNA generally varies from one species to another.

2. DNA specimens isolated from different tissues of the same species have the same base composition.

3. The base composition of DNA in a given species does not change with an organism’s age, nutritional state, or changing environment.

4. In all cellular , regardless of the species, the

– A= T & G=C.

– A+G = T+ C. PURINE=PYRIMIINE Chemical Structure of DNA vs RNA have a 2’-OH Deoxyribonucleotides have a 2’-H Nucleotides and Nucleosides

• Nucleotides are the monomeric constituents of DNA and RNA. They are composed of; – 5-C Sugar – Nitrogenous base – Phosphate group

• Nucleosides are composed of; – A sugar – Nitrogenous base

Nucleosides vs. Nucleotides Nucleotides, cont’d

• Nucleotides are joined (polymerized) by condensation reactions to form chains of DNA and RNA

• The –OH on the 3’-C of a sugar of one nucleotide forms an ester bond to the phosphate at 5’-C of another nucleotide;

– The 5’ – 3’ Phosphodiester bond

The Phosphodiester bond Nucleotides are linked by phosphodiester bonds Significance of 5’ and 3’ • The two ends of DNA/RNA are designated by symbols 5’ and 3’ – 5’ refers to C in the sugar to which a phosphate (PO4) functional group is attached

– 3’ refers to C in the sugar to which a –OH functional group is attached

– Thus, symmetry of the end of a DNA strand implies that each strand has a polarity determined by which end bears the 5’-PO4 and which end bears the 3’-OH group Significance of 5’ and 3’

• 5’ – 3’ directionality. Understanding this polarity is critical for understanding;

– DNA replication

– Transcription

– Reading DNA/RNA sequences

– Carrying out an experiment in a lab

• DNA sequences are written with the 5’-end to the left and 3’-end to the right;

– 5’-TGGCCCGGGTCGACGGTGACACCGTGTTC-3’ Nomenclature of Nucleotides

-The triphosphate form is the precursor building block for DNA and RNA chain Secondary structure of DNA: the Double Helix

• DNA is a double helix;

• Characterized with H-bonding between bases of two strands

– H-bonding also referred to as “Watson-Crick” or “Complementary base pairing”

– A with T, two H-bonds; G with C, three H-bonds

– 1’-C atoms in the two strands are exactly the same distance apart (1.08 nm)

– Watson-Crick base pairing explains Chargaff’s rules Structure of Watson-Crick double helix 1. Alternating sugars and PO4 groups form the backbone of DNA 2. Bases are attached to sugars, located between the backbones of the DNA strands, lying perpendicular to the long axis of the strands 3. As backbones of the two strands wind around each other, they form a double helix 4. Polarity in each strand is 5’ to 3’ of the double helix; one end has a 5’-PO4, the other end 3’-OH 5. H-bonding occurs only if polarity of the two strands runs in opposite direction; hence the two strands of the double helix are antiparallel 6. Double helix has major and minor grooves The double helix, cont’d DNA can undergo reversible strand separation

• Reversible strand separation allows DNA replication, Transcription, Translation with high fidelity

• Same features make it possible to manipulate DNA in vitro – Unwinding and separation of DNA strands is referred to as denaturation – The temperature at which half the bases in a DNA sample have denatured is called melting temperature (Tm) Denaturing, Renaturing, Hybridization of DNA Ribonucleic acid (RNA): types & structure • Polymer of nucleotides – What is the difference? – Single strandeD – OH at C-2

• Five RNA types: – rRNA – mRNA – tRNA Components of RNA The versatility of RNA • Folds into unique 3-D structures which act similarly to globular proteins; “tRNA looks like natures attempt to make RNA do the job of a ” -

• Generally the pathway of expression from DNA to functional product via an RNA intermediate overemphasizes proteins as the ultimate goal; • However, RNAs are involved in a variety of cellular processes along the pathway of gene expression including; – DNA replication; RNA processing; mRNA turnover; protein synthesis; protein targeting

• RNA catalyzes chemical reactions in living cells (ribozymes) tRNA • Mainly found in cytoplasm

• Single stranded made up of up to 75 nucleotides.

• Their bases include adenine, guanine, cytosine, uracil and pseudouridine.

• Each single strand of tRNA is folded to form a clover leaf structure. tRNA

Adaptor like molecule that decodes mRNA into amino acids

Brings amino acid corresponding to the appropriate mRNA codon

Each amino acid has unique tRNA tRNA structure

The three tRNA loops form the cloverleaf secondary structure; each loop has a specific function

1. The T-loop; involved in recognition by the ribosomes

2. The D-loop; is associated with recognition by the aminoacyl tRNA synthatase

3. The anticodon loop: base pairs with the codon in mRNA • Every tRNA has the sequence ACC on the 3’-End to which the amino acid is attached

• The anticodon loop in all tRNAs is bounded by U on the 5’- side and a modified purine on the 3’-side tRNA: secondary and tertiary structures rRNA

• Component of the ribosome, the site of protein synthesis in all living cells • Provides a mechanism for decoding mRNA into amino acids and interacts with tRNAs during translation by providing peptidyltransferase activity

Type Size Large subunit Small subunit

prokaryotic 70S 50S (5S, 23S) 30S (16S)

eukaryotic 80S 60S (5S, 5.8S, 28S) 40S (18S)

NOTE: -Sites of attack for several antimicrobials -DRUG TARGETS -used for classification of microorganisms -TYPING Ribonucleoproteins (RNPs)

• Most RNAs are associated with proteins as RNA-protein complexes called ribonucleotpoteins – One important RNP is the ribosome

• RNA-based catalytic reactions occur in conjunction with proteins

• Catalytic RNAs are called ribozymes; they catalyze a number of reactions in cells ranging from cleavage of phosphodiester bonds to peptide formation mRNA

Information in DNA copied (transcribed) into mRNA, which goes to ribosome for translation -unstable in cells (why?), <5% total RNA -Methylated heads (CH3 cup) -Polyadenylated tails: utilized in biotech -Represents expressed genes in cells mRNA

• Consists of A, G, C and U as the major bases and methyl purines and methyl pyrimidines as minor bases.

• The mRNA molecules are formed with the help of DNA template strand during the process of transcription

• The mRNA carries a specific sequence of nucleotides in triplets called codons responsible for the synthesis of a specific protein molecule.

mRNA • The 3’-OH end of most mRNA carries a polymer of adenylate ribonucleotides consisting to 20 to 250 residues, called the poly A tail.

• The poly A tail maintains the intacellular stability of the specific mRNA by preventing the attack of 3’- exonucleases. mRNA

• The 5’OH end of the mRNA carries a cap structure consisting of 7-methyl GTP.

• This cap is involved in recognition of protein biosynthetic machinery and it helps in stabilizing the mRNA by preventing the attack by 5’-exonucleases.

• Protein synthesis begins at the 5’ end of the capped structure of mRNA. Relationship between the 5 RNA types during gene expression Summary 1. DNA and RNA are chain-lie molecules composed of subunits called nucleotides 2. Nucleotides contain a base linked to the 1’-position of a sugar and a phosphate group 3. Phosphate joins the sugars in a DNA or RNA chain through their 5’- and 3’-hydroxyl groups by phosphodiester 4. The DNA molecule is a double helix, with sugar-phosphate backbones on the outside and base pairs on the inside 5. The bases pair in a specific way: – Adenine (A) with thymine (T) – Guanine (G) with cytosine (C) • bonds

2-47 Biomedical importance

• At neutral pH, guanine is the least soluble of the bases followed by xanthine.

• Although uric acid, as urate is relatively soluble at neutral pH, it is highly insoluble in solutions with a lower pH, such as urine.

• Xanthine and uric acid which are constituents of human urine may form urinary tract stones as xanthine stone and/or urate stones. Unusual bases

• May be found in DNA and tRNA of both prokaryotes and eukaryotes e.g.

• N6-methyl adenine, N7 methyl guanine and N6-N6- dimethyl adenine found in mRNA molecules.

• 5-methylcytosine found in both bacterial and human DNA

Bases in plants

• Some purine bases found in plants have pharmacologic properties: • Theophylline: 1,3-dimethylxanthine is found in tea • Theobromine: 3, 7 dimethyl xanthine found in cocoa • Caffeine present in coffee is 1, 3, 7-trimethyl xanthine. Synthetic analogues of Biomedical Importance

• In these compounds, the heterocyclic ring structure or the sugar moiety is altered in such a way as to induce toxic effects when the analogues get incorporated into cellular constituents of the body.

• Effects result from either: – Inhibition of specific by the drug – Incorporation of metabolites of the drug into the nucleic acids where they adversely affect the structure.

Synthetic derivatives

• Anticancer agents such as 6-thio guanine, 6- mercaptopurine and Azapurine

• Allopurinol which inhibits uric acid formation

• Cytarabine and vidarabine, contain the sugar arabinose in place of used as anticancer and antiviral agents

• Aminophylline and theophylline used to inhibit the catabolism of cAMP. Biological importance • ATP: – "molecular unit of currency" of intracellular energy transfer.

– ATP is used as a substrate in signal transduction pathways by kinases that phosphorylate proteins and lipids.

– Adenylate cyclase, which uses ATP to produce the second messenger molecule cyclic AMP.

Biological importance • ADP – ADP-ATP cycling supplies the energy needed to do work in a biological system

– Substrate for glycolysis, TCA cycle, oxidative phosphorylation

– Involved in blood platelet activation

• AMP – Involved in formation of cAMP – Regulation of metabolic pathways

Biological importance • cAMP – Second messenger, Derived from ATP

– used for intracellular signal transduction, such as transferring into cells the effects of hormones like glucagon and adrenaline, which cannot pass through the plasma membrane.

– It is involved in the activation of protein kinases and regulates the effects of adrenaline and glucagon. Biological importance • GTP – GTP is involved in energy transfer within the cell e.g. source of energy for protein synthesis and gluconeogenesis.

– During the process of translation, GTP is used as an energy source

– substrate for the synthesis of RNA and DNA

– Involved in microtubule polymerization

Biological importance • UDP – UDP glucose • Involved in glycogen metabolism • precursor of sucrose lipopolysaccharides, and glycosphingolipids. – UDP-glucuronic acid • Conjugation and detoxification reactions in liver • Synthesis of hyaluronic acid and heparin – UDP-galacturonic acid and UDP-L-iduronic acids for synthesis of chondritin sulphate

Biological importance • Cytidine nucleotides – CMP is used to activate mannose in metabolism.

– CDP-choline- intermmediate in synthesis of phosphatidyl choline

– CDP-glycerol involved in biosynthesis of teichoic acid in bacteria

– CDP-ethanolamine pathways are used to synthesize phosphatidyl ethanolamine

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