DNA & RNA

DNA and RNA Overview

  • DNA and RNA serve as the basis of genetic material in all life forms.

  • Composed of monomeric units called nucleotides.

  • Components of nucleotides:

    • Nitrogenous base (determines coding identity)

    • Five-carbon carbohydrate (sugar; aldopentose)

    • One, two, or three phosphate groups.

Types of Nitrogenous Bases

  • Two categories:

    • Purines: Adenine, Guanine

    • Pyrimidines: Cytosine, Thymine (DNA), Uracil (RNA).

  • Structure of purines and pyrimidines indicated alongside their chemical formulas.

Sugar and Phosphate in Nucleotides

  • The ribose sugar and its variability:

    • 2’ position can have either an OH group (in RNA) or H (in DNA).

  • Glycosidic bonds:

    • Form to N9 in purines and N1 in pyrimidines.

  • Carbon atoms in ribose are marked with a prime symbol to avoid confusion with base atoms (e.g., 5’ vs. 5).

Sugar Conformation in DNA and RNA

  • Ribose (an aldose) cyclizes into:

    • β-D-furanose in RNA

    • β-2’-deoxy-D-furanose in DNA.

  • Glycosidic bond formation locks conformation in place.

  • Furanose adopts two puckered forms (C-2’ endo in B-form DNA and C-3’ endo in RNA).

Nucleotide and Nucleic Acid Nomenclature

  • Purines:

    • Adenine ➔ Adenosine ➔ Adenylate (RNA)

    • Deoxyadenosine ➔ Deoxyadenylate (DNA)

    • Guanine ➔ Guanosine ➔ Guanylate (RNA)

    • Deoxyguanosine ➔ Deoxyguanylate (DNA)

  • Pyrimidines:

    • Cytosine ➔ Cytidine ➔ Cytidylate (RNA)

    • Deoxycytidine ➔ Deoxycytidylate (DNA)

    • Thymine ➔ Thymidine ➔ Thymidylate (DNA)

    • Uracil ➔ Uridine ➔ Uridylate (RNA).

  • Nomenclature encompasses both ribo- and deoxyribo- forms.

Phosphate Groups in Nucleotides

  • Nucleotides may have one, two, or three phosphate groups attached to the 5’ carbon of the sugar.

  • Phosphate groups are designated as α, β, or γ.

Formation of Polynucleotides

  • Polynucleotides (DNA and RNA) have directional ends (5’ end and 3’ end).

  • Nucleotides are linked via phosphodiester bonds.

  • The 5’ end contains phosphates, while the 3’ end has hydroxyl groups.

  • Polynucleotide synthesis involves adding NTPs or dNTPs to the 3’ end.

Characteristics of DNA and RNA Helices

  • DNA and RNA strands form anti-parallel double helices.

  • Base pairing:

    • Adenine pairs with Thymine (or Uracil in RNA, via 2 H-bonds)

    • Cytosine pairs with Guanine (via 3 H-bonds, offering more stability).

  • Sugars and phosphodiester backbone remain unchanged; only bases differ.

Identifying the 2' OH Group

  • Visual identification of molecular models highlighting the position of the 2’ hydroxyl group.

Structural Features of Nucleic Acids

  • Phosphate backbone:

    • Highly acidic, favors interaction with water.

  • Nitrogenous bases:

    • Uncharged and hydrophobic, minimizing exposure to water through stacking.

  • Hydrogen bonding occurs between bases (G⟷C, A⟷T/U) facilitating stable double helix structures.

Double Helix Formation

  • Oligonucleotide strands form a double helix when base sequences are complementary.

  • Helix stabilization is through stacking and hydrogen bond interactions.

  • Strands are anti-parallel, aligning major and minor grooves for accessibility.

Base Pair Interactions

  • Major groove contacts bases on the Watson-Crick face facilitating interactions.

  • Minor groove offers different access to base pairs.

Forms of Nucleic Acids

  • Types: A form (for RNA) and B form (for DNA).

  • The presence of 2’OH in RNA versus its absence in DNA differentiates their conformations.

Historical Evidence for DNA as Genetic Material

  • Transformation agent identified as DNA via experiments by Avery, McLeod, and McCarthy.

  • Hershey-Chase Experiment corroborated DNA's role as genetic material.

DNA Denaturation and Renaturation

  • Heating doublestranded DNA causes hydrogen bond breakage, leading to denaturation.

  • DNA can renature upon slow cooling.

  • Absorption characteristics: DNA and RNA absorb light at 260 nm, with melting causing increased absorbance.

RNAs Secondary Structure

  • While DNA is rigid, the 2’OH in RNA introduces flexibility to form structures like bends, loops, and hairpins.

  • Watson-Crick pairing rules apply (A⟷U).

Classes of RNA - tRNAs

  • tRNAs are smallest RNA types, consisting of single strands that fold into common shapes despite sequence variations.

Classes of RNA - rRNAs

  • rRNAs (23S, 16S, 5S in prokaryotes) are larger and adopt shapes crucial for ribosome functionality.

  • Distinct regions are conserved across kingdoms, whereas some exhibit divergence.

Nucleases Classifications

  • Nucleases hydrolyze phosphodiester bonds:

    • Deoxyribonucleases (DNases)

    • Ribonucleases (RNases)

    • Exonucleases (degrade from ends)

    • Endonucleases (internal cleavage).

Endonuclease Functions

  • Endonucleases cleave internal phosphodiester bonds.

  • Restriction endonucleases recognize palindromic sequences for cleaving chromosomal DNA.

  • Recognition sites and cut sequences of common restriction endonucleases listed in a table format.

Identifying Restriction Endonuclease Sites

  • Question: Which sequence could potentially be a restriction endonuclease site?

    • Options:

    • a) 5’ GGGCCC 3’

    • b) 5’ GAGAGA 3’

    • c) 5’ AAAAAA 3’

    • d) 5’ GTCGTC 3’

    • e) 5’ FATCAT 3’

Origin of Restriction Endonucleases

  • Bacteria produce restriction endonucleases as a defense against bacteriophages.

  • Host methylases recognize and methylate DNA sequences, protecting them from cleavage.

  • Phage DNA remains unmodified and is degraded by bacterial endonucleases.

DNA Organization in Chromatin

  • DNA organized into chromatin through interactions with proteins.

  • Smallest chromatin unit = nucleosome, composed of histone proteins (2 x H2A, H2B, H3, H4).

  • Nucleosomes create condensed structures termed "beads on a string".

Reiteration of DNA Organization

  • Same details as Page 26 regarding chromatin structure and nucleosome formation.

DNA Replication and Repair

  • The DNA sequence is the basis for genetic information storage.

  • Replication required when a parent cell divides, necessitating synthesis from the parental template.

  • Initial studies on DNA replication were primarily conducted in E. coli.

Models of DNA Replication

  • Three potential replication models:

    • DNA replication could be conservative, semi-conservative, or dispersive.

  • Evidence supports the semi-conservative model.

DNA Density Gradient Centrifugation

  • Procedure details for density gradient centrifugation:

    • DNA mixed with CsCl, followed by high-speed centrifugation forming a density gradient.

  • DNA separates at levels matching its density.

Heavier Isotope DNA in Centrifugation

  • DNA from heavy isotopes sediments deeper than that from light isotopes.

  • The semi-conservative nature of DNA replication is demonstrated through generation observations.

Complementary DNA Sequence

  • Inquiry for the complementary sequence to DNA 5’ GTCTTGCATG 3’, reminding to write from 5’ to 3’ direction.

DNA Replication Process Overview

  • DNA replication begins at specific origins, forming bubbles and forks as daughter duplexes emerge.

DNA Replication Observation Techniques

  • The replication initiation is visually observed using 3H-thymidine, appearing on photographic film like an X-ray.