Nucleic Acids Nucleic Acids Are Essential For Information

  • Slides: 56
Download presentation
Nucleic Acids

Nucleic Acids

Nucleic Acids Are Essential For Information Transfer in Cells • Information encoded in a

Nucleic Acids Are Essential For Information Transfer in Cells • Information encoded in a DNA molecule is transcribed via synthesis of an RNA molecule • The sequence of the RNA molecule is "read" and is translated into the sequence of amino acids in a protein.

Central Dogma of Biology

Central Dogma of Biology

Nucleic Acids • First discovered in 1869 by Miescher. • Found as a precipitate

Nucleic Acids • First discovered in 1869 by Miescher. • Found as a precipitate that formed when extracts from nuclei were treated with acid. • Compound contained C, N, O, and high amount of P. • Was an acid compound found in nuclei therefore named nucleic acid

Nucleic Acids • 1944 Oswald, Avery, Mac. Leod and Mc. Carty demonstrated that DNA

Nucleic Acids • 1944 Oswald, Avery, Mac. Leod and Mc. Carty demonstrated that DNA is the molecule that carrier genetic information. • 1953 Watson and Crick proposed the double helix model for the structure of DNA

Nucleic Acids • Nucleic acids are long polymers of nucleotides. • Nucleotides contain a

Nucleic Acids • Nucleic acids are long polymers of nucleotides. • Nucleotides contain a 5 carbon sugar, a weakly basic nitrogenous compound (base), one or more phosphate groups. • Nucleosides are similar to nucleotides but have no phosphate groups.

Pentoses of Nucleotides • D-ribose (in RNA) • 2 -deoxy-D-ribose (in DNA) • The

Pentoses of Nucleotides • D-ribose (in RNA) • 2 -deoxy-D-ribose (in DNA) • The difference - 2'OH vs 2'-H • This difference affects secondary structure and stability

Nitrogenous Bases

Nitrogenous Bases

Bases are attached by b-Nglycosidic linkages to 1 carbon of pentose sugar – (Nucleoside)

Bases are attached by b-Nglycosidic linkages to 1 carbon of pentose sugar – (Nucleoside)

Nucleosides • Base is linked via a b-Nglycosidic bond • The carbon of the

Nucleosides • Base is linked via a b-Nglycosidic bond • The carbon of the glycosidic bond is anomeric • Named by adding -idine to the root name of a pyrimidine or -osine to the root name of a purine • Conformation can be syn or anti • Sugars make nucleosides more water-soluble than free bases

Anti- conformation predominates in nucleic acid polymers

Anti- conformation predominates in nucleic acid polymers

Nucleotides • Phosphate ester of nucleosides

Nucleotides • Phosphate ester of nucleosides

The plane of the base is oriented perpendicular to the plane of the pentose

The plane of the base is oriented perpendicular to the plane of the pentose group

Other Functions of Nucleotides • Nucleoside 5'-triphosphates are carriers of energy • Bases serve

Other Functions of Nucleotides • Nucleoside 5'-triphosphates are carriers of energy • Bases serve as recognition units • Cyclic nucleotides are signal molecules and regulators of cellular metabolism and reproduction • ATP is central to energy metabolism • GTP drives protein synthesis • CTP drives lipid synthesis • UTP drives carbohydrate metabolism

 • Nucleotide monomers are joined by 3’-5’ phosphodiester linkages to form nucleic acid

• Nucleotide monomers are joined by 3’-5’ phosphodiester linkages to form nucleic acid (polynucleotide) polymers

Nucleic Acids • Nucleic acid backbone takes on extended conformation. • Nucleotide residues are

Nucleic Acids • Nucleic acid backbone takes on extended conformation. • Nucleotide residues are all oriented in the same direction (5’ to 3’) giving the polymer directionality. • The sequence of DNA molecules is always read in the 5’ to 3’ direction

Bases from two adjacent DNA strands can hydrogen bond • Guanine pairs with cytosine

Bases from two adjacent DNA strands can hydrogen bond • Guanine pairs with cytosine • Adenine pairs with thymine

Base pairing evident in DNA compositions

Base pairing evident in DNA compositions

H-bonding of adjacent antiparallel DNA strands form double helix structure

H-bonding of adjacent antiparallel DNA strands form double helix structure

Properties of DNA Double Helix • Distance between the 2 sugar-phosphate backbones is always

Properties of DNA Double Helix • Distance between the 2 sugar-phosphate backbones is always the same, give DNA molecule a regular shape. • Plane of bases are oriented perpendicular to backbone • Hydrophillic sugar phosphate backbone winds around outside of helix • Noncovalent interactions between upper and lower surfaces of base-pairs (stacking) forms a closely packed hydrophobic interior. • Hydrophobic environment makes H-bonding between bases stronger (no competition with water) • Cause the sugar-phosphate backbone to twist.

View down the Double Helix Hydrophobic Interior with base pair stacking Sugar-phosphate backbone

View down the Double Helix Hydrophobic Interior with base pair stacking Sugar-phosphate backbone

Structure of DNA Double Helix • Right handed helix • Rise = 0. 33

Structure of DNA Double Helix • Right handed helix • Rise = 0. 33 nm/nucleotide • Pitch = 3. 4 nm / turn • 10. 4 nucleotides per turn • Two groves – major and minor

 • Within groves, functional groups on the edge of base pairs exposed to

• Within groves, functional groups on the edge of base pairs exposed to exterior • involved in interaction with proteins.

Factors stabilizing DNA double Helix • Hydrophobic interactions – burying hydrophobic purine and pyrimidine

Factors stabilizing DNA double Helix • Hydrophobic interactions – burying hydrophobic purine and pyrimidine rings in interior • Stacking interactions – van der Waals interactions between stacked bases. • Hydrogen Bonding – H-bonding between bases • Charge-Charge Interactions – Electrostatic repulsions of negatively charged phosphate groups are minimized by interaction with cations (e. g. Mg 2+)

DNA • 1 o Structure - Linear array of nucleotides • 2 o Structure

DNA • 1 o Structure - Linear array of nucleotides • 2 o Structure – double helix • 3 o Structure - Super-coiling, stemloop formation • 4 o Structure – Packaging into chromatin

Determination of the DNA 1 o Structure (DNA Sequencing) • Can determine the sequence

Determination of the DNA 1 o Structure (DNA Sequencing) • Can determine the sequence of DNA base pairs in any DNA molecule • Chain-termination method developed by Sanger • Involves in vitro replication of target DNA • Technology led to the sequencing of the human genome

DNA Replication • DNA is a double-helical molecule • Each strand of the helix

DNA Replication • DNA is a double-helical molecule • Each strand of the helix must be copied in complementary fashion by DNA polymerase • Each strand is a template for copying • DNA polymerase requires template and primer • Primer: an oligonucleotide that pairs with the end of the template molecule to form ds. DNA • DNA polymerases add nucleotides in 5'-3' direction

Chain Termination Method • Based on DNA polymerase reaction • 4 separate rxns •

Chain Termination Method • Based on DNA polymerase reaction • 4 separate rxns • Each reaction mixture contains d. ATP, d. GTP, d. CTP and d. TTP • Each reaction also contains a small amount of one dideoxynucleotide (dd. ATP, dd. GTP, dd. CTP and dd. TTP). • Each of the 4 dideoxynucleotides are labeled with a different fluorescent dye. • Dideoxynucleotides missing 3’-OH group. Once incorporated into the DNA chain, chain elongation stops)

Chain Termination Method • Most of the time, the polymerase uses normal nucleotides and

Chain Termination Method • Most of the time, the polymerase uses normal nucleotides and DNA molecules grow normally • Occasionally, the polymerase uses a dideoxynucleotide, which adds to the chain and then prevents further growth in that molecule • Random insertion of dd-nucleotides leaves (optimally) at least a few chains terminated at every occurrence of a given nucleotide

Chain Termination Method • Run each reaction mixture on electrophoresis gel • Short fragments

Chain Termination Method • Run each reaction mixture on electrophoresis gel • Short fragments go to bottom, long fragments on top • Read the "sequence" from bottom of gel to top • Convert this "sequence" to the complementary sequence • Now read from the other end and you have the sequence you wanted - read 5' to 3'

DNA Secondary structure • DNA is double stranded with antiparallel strands • Right hand

DNA Secondary structure • DNA is double stranded with antiparallel strands • Right hand double helix • Three different helical forms (A, B and Z DNA.

Comparison of A, B, Z DNA • A: right-handed, short and broad, 2. 3

Comparison of A, B, Z DNA • A: right-handed, short and broad, 2. 3 A, 11 bp per turn • B: right-handed, longer, thinner, 3. 32 A, 10 bp per turn • Z: left-handed, longest, thinnest, 3. 8 A, 12 bp per turn

A-DNA B-DNA Z-DNA

A-DNA B-DNA Z-DNA

Z-DNA • Found in G: Crich regions of DNA • G goes to syn

Z-DNA • Found in G: Crich regions of DNA • G goes to syn conformation • C stays anti but whole C nucleoside (base and sugar) flips 180 degrees

DNA sequence Determines Melting Point • Double Strand DNA can be denatured by heat

DNA sequence Determines Melting Point • Double Strand DNA can be denatured by heat (get strand separation) • Can determine degree of denturation by measuring absorbance at 260 nm. • Conjugated double bonds in bases absorb light at 260 nm. • Base stacking causes less absorbance. • Increased single strandedness causes increase in absorbance

DNA sequence Determines Melting Point • Melting temperature related to G: C and A:

DNA sequence Determines Melting Point • Melting temperature related to G: C and A: T content. • 3 H-bonds of G: C pair require higher temperatures to denture than 2 Hbonds of A: T pair.

DNA o 3 Structure • Super coiling • Cruciform structures

DNA o 3 Structure • Super coiling • Cruciform structures

Supercoils • In duplex DNA, ten bp per turn of helix (relaxed form) •

Supercoils • In duplex DNA, ten bp per turn of helix (relaxed form) • DNA helix can be over-wound. • Over winding of DNA helix can be compensated by supercoiling. • Supercoiling prevalent in circular DNA molecules and within local regions of long linear DNA strands • Enzymes called topoisomerases or gyrases can introduce or remove supercoils • In vivo most DNA is negatively supercoiled. • Therefore, it is easy to unwind short regions of the molecule to allow access for enzymes

Each super coil compensates for one + or – turn of the double helix

Each super coil compensates for one + or – turn of the double helix

 • Cruciforms occur in palindromic regions of DNA • Can form intrachain base

• Cruciforms occur in palindromic regions of DNA • Can form intrachain base pairing • Negative supercoiling may promote cruciforms

DNA o 4 Structure • In chromosomes, DNA is tightly associated with proteins

DNA o 4 Structure • In chromosomes, DNA is tightly associated with proteins

Chromosome Structure • Human DNA’s total length is ~2 meters! • This must be

Chromosome Structure • Human DNA’s total length is ~2 meters! • This must be packaged into a nucleus that is about 5 micrometers in diameter • This represents a compression of more than 100, 000! • It is made possible by wrapping the DNA around protein spools called nucleosomes and then packing these in helical filaments

Nucleosome Structure • Chromatin, the nucleoprotein complex, consists of histones and nonhistone chromosomal proteins

Nucleosome Structure • Chromatin, the nucleoprotein complex, consists of histones and nonhistone chromosomal proteins • % major histone proteins: H 1, H 2 A, H 2 B, H 3 and H 4 • Histone octamers are major part of the “protein spools” • Nonhistone proteins are regulators of gene expression

 • 4 major histone (H 2 A, H 2 B, H 3, H

• 4 major histone (H 2 A, H 2 B, H 3, H 4) proteins for octomer • 200 base pair long DNA strand winds around the octomer • 146 base pair DNA “spacer separates individual nucleosomes • H 1 protein involved in higher-order chromatin structure. • W/O H 1, Chromatin looks like beads on string

Solenoid Structure of Chromatin

Solenoid Structure of Chromatin

RNA • Single stranded molecule • Chemically less stable than DNA • presence of

RNA • Single stranded molecule • Chemically less stable than DNA • presence of 2’-OH makes RNA more susceptible to hydrolytic attack (especially form bases) • Prone to degradation by Ribonucleases (Rnases) • Has secondary structure. Can form intrachain base pairing (i. e. cruciform structures). • Multiple functions

Type of RNA • Ribosomal RNA (r. RNA) – integral part of ribosomes (very

Type of RNA • Ribosomal RNA (r. RNA) – integral part of ribosomes (very abundant) • Transfer RNA (t. RNA) – carries activated amino acids to ribosomes. • Messenger RNA (m. RNA) – endcodes sequences of amino acids in proteins. • Catalytic RNA (Ribozymes) – catalzye cleavage of specific RNA species.

RNA can have extensive 2 o structure

RNA can have extensive 2 o structure