Molecular Structure of DNA and RNA Notes

Molecular Structure of DNA and RNA

Genetic Material Criteria

  • Genetic material must meet several criteria:
    • Information: It must contain the information necessary to make an entire organism.
    • Transmission: It must be passed from parent to offspring.
    • Replication: It must be copied in order to be passed from parent to offspring.
    • Variation: It must be capable of changes to account for the known phenotypic variation in each species.

DNA and RNA Structure

  • DNA and RNA are large macromolecules with several levels of complexity.
  • Nucleotides form the repeating unit of nucleic acids.
  • In a DNA molecule, two strands interact to form a double helix.
  • The 3-D structure of DNA results from folding and bending of the double helix.
  • Interaction of DNA with proteins produces chromosomes within living cells.

Components of Nucleotides

  • The nucleotide is the repeating structural unit of DNA and RNA.
  • It has three components:
    • A phosphate group
    • A pentose sugar
      • Ribose in RNA
      • Deoxyribose in DNA
    • A nitrogenous (nitrogen-containing) base
      • Purines: A (Adenine), G (Guanine)
      • Pyrimidines: T (Thymine), C (Cytosine), U (Uracil, in RNA)

Structure of Nucleotides in DNA and RNA

  • (a) Repeating unit of deoxyribonucleic acid (DNA).
  • (b) Repeating unit of ribonucleic acid (RNA).
  • Carbon numbering is important:
    • C1': base is attached here
    • C2': -H for Deoxyribose/DNA; -OH for Ribose/RNA
    • C3': -OH (connect to C5' of another nucleotide via a phosphate group)
    • C5': phosphate group attached (connects to C3' of another nucleotide via a phosphate group)

Nucleotide Linkage

  • Nucleotides are linked by covalent ester bonds.
  • Phosphodiester Bond: bond linking two sugars at the C3' of one sugar and the C5' of another sugar.
  • Forms the sugar-phosphate backbone of the DNA strand.
  • Polarity (directionality): 5' (free phosphate) to 3' (free hydroxyl group).
  • DNA sequence: 5' to 3'; nucleotides are added to the 3' end.

DNA Strand Directionality

  • A DNA strand has 5' to 3' directionality.
  • In a strand, all sugar molecules are oriented in the same direction.
  • Directionality is based on the orientation of the sugar molecule within the strand.
  • The phosphates and sugar molecules form the backbone of the nucleic acid strand.
  • The bases project from the backbone.

DNA Double Helix

  • Double helix: Two single strands of DNA running antiparallel.
  • Sequences are complementary but not the same.
  • The double-helical structure of DNA is stabilized by:
    • Hydrogen bonding between complementary bases
      • A bonded to T by two hydrogen bonds
      • C bonded to G by three hydrogen bonds

Chargaff’s Rule

  • Rules of base pairing:
    • Hundreds of measurements were made, and the compelling observation was that:
      • Percent of adenine = percent of thymine
      • Percent of cytosine = percent of guanine
    • This observation became known as Chargaff’s rule.
    • It was a crucial piece of evidence that Watson and Crick used to elucidate the structure of DNA.

DNA Double Helix Stabilization

  • Two types of forces to stabilize the double helix:
    • Within a base pair: Hydrogen bonds between complementary bases: A = T; C ≡ G
    • Between base pairs: Base stacking force
      • A kind of hydrophobic force: the tendency of hydrophobic bases to stack with each other to avoid exposure to the water environment.
      • Major contributor to double helix stability.

Structure of the DNA Double Helix: Key Features

  • Two strands of DNA form a right-handed double helix.
  • Bases in opposite strands hydrogen bond according to the AT/GC rule.
  • The two strands are antiparallel, one runs in the 5' to 3' direction, and the other 3' to 5'.
  • Approximately 10.0 nucleotides in each strand per complete 360° turn of the helix.
  • The helix is right-handed; as it spirals away, it turns in a clockwise direction.

Protein Binding at Major and Minor Grooves

  • Two asymmetrical grooves on the outside of the helix:
    • Major groove
    • Minor groove
  • Grooves create the site of attachment of DNA-binding proteins that are involved in the process of replication and transcription.
  • Major groove: wider, more exposed; exposed bases have unique patterns recognizable by proteins.
  • Minor groove: narrower, bases are less exposed.

DNA-Protein Interaction

  • Certain proteins can bind within these grooves.
  • These proteins recognize and bind to specific molecular information displayed at the edges of base pairs and accessible in the grooves.
  • Slight bends, local irregularities, and other variations result in regions of the double helix with slight distortions; these areas can be recognized by proteins.

Sequence Specificity of DNA Binding Protein

  • Specific interactions between macromolecules are like a key and lock – two molecules physically fit with each other.
  • Interactions between negatively charged DNA backbone and positively charged proteins (histone) are non-sequence-specific interactions.
  • Interactions between grooves and transcription factors are often sequence-specific.

DNA Sequence Recognition

  • Direct readout: Protein binds directly to specific base pairs on the DNA (sequence recognition by the protein).
    • Different base pairs --> different shapes of the groove surfaces --> fit by different proteins that bind to grooves.
  • Indirect readout: The protein recognizes the DNA sequence not by directly contacting the base pairs themselves, but by sensing the structural features of the DNA backbone, like its flexibility or curvature, which are influenced by the sequence, allowing the protein to identify specific DNA regions without making direct base contacts (structural recognition).
    • Different base pairs --> different backbone shapes --> fit by different proteins that bind to the backbone.

DNA Denaturation and Renaturation

  • Hydrogen bonds between bases and base stacking are non-covalent bonds, so it is possible to separate and reform the double helix.
  • Heating provides enough energy to separate the strands.
  • Separation of the two strands is called denaturing or melting.
  • Complementary strands can renature/anneal/hybridize when the temperature is decreased again.

Melting Temperature (Tm)

  • Tm = melting temperature: Defined as the temperature when half of the helices are separated.
  • Tm is determined by:
    1. DNA length (both base stacking force and hydrogen bonds)
    2. G/C content - Not all nucleic acids have the same G/C content, can vary from 26 to 74%.
      • A = T (2 hydrogen bonds)
      • C ≡ G (3 hydrogen bonds) --> A-T bonds denature easier than G-C bonds
      • Higher G/C content --> higher Tm

Tm and Molecular Biology Techniques

  • Tm = melting temperature: Critical factor in many molecular biology techniques including PCR, Southern blotting, and DNA hybridizations.
  • PCR machine: thermal cycler, repeated denature and renature
  • An important parameter of PCR: Tm
    • TaOpt=0.3×(Tm of primer)+0.7×(Tm of product)–14.9Ta_{Opt} = 0.3 \times (Tm \text{ of primer}) + 0.7 \times (Tm \text{ of product}) – 14.9

Polymerase Chain Reaction (PCR)

  • PCR is a way to copy a specific sequence of DNA.
  • Developed by Kary Mullis in 1985.
  • PCR can copy DNA without the aid of vectors and host cells.
  • Must know enough about the gene of interest to have the sequence of two short primers.
  • Melting temperature (Tm) is important for PCR because it helps determine the thermal cycling parameters and the annealing temperature.

Starting Material for PCR

  1. Template DNA: Contains the region that needs to be amplified.
  2. Oligonucleotide primers:
    • Complementary to sequences at the ends of the DNA fragment to be amplified.
    • These are synthetic and about 15 to 20 nucleotides long.
  3. Deoxynucleoside triphosphates (dNTPs): Provide the precursors for DNA synthesis.
  4. Taq polymerase:
    • DNA polymerase isolated from the bacterium Thermus aquaticus.
    • This thermostable enzyme is necessary because PCR involves heating steps that inactivate most other DNA polymerases.

Steps of a PCR Cycle

  • One PCR cycle includes three steps:
    1. Denaturation: DNA strands are separated.
    2. Primer annealing: Oligonucleotide primers bind to the DNA strands.
    3. Primer extension: Nucleotides are added to the primers, thereby extending their lengths.
  • Repeated cycles will produce thousands of copies of the region of interest, even from a large background of other sequences.

PCR Amplification

  • PCR is carried out in a thermocycler; the sequential process of denaturing-annealing-synthesis is then repeated for many cycles (20–30 cycles).
  • After 20 cycles, a target DNA sequence will increase 2202^{20}-fold (~1 million-fold).
  • After 30 cycles, a target DNA sequence will increase 2302^{30}-fold (~1 billion-fold).
  • This assumes 100% efficiency.

Uses of PCR

  • A specific DNA segment can be amplified from a complex mixture of other sequences (one gene out of an entire genome).
  • PCR can also be used to amplify chromosomal DNA non-specifically.
    • A mixture of primers with many different random sequences is used.
    • These will anneal randomly throughout the genome and amplify most of the chromosomal DNA.
  • Used to amplify very small samples, such as DNA from crime scenes.

Denaturation/Renaturation (Hybridization)

  • Can occur between:
    • i. DNA-DNA strands
    • ii. DNA-RNA strands
    • iii. RNA-RNA strands (intermolecular pairing between RNAs)
    • iv. RNA-RNA strands (intramolecular pairing within RNA)
  • Renaturation only occurs between molecules of complementary sequences.
  • Allows precise detection of specific DNA/RNA sequences (e.g., CRISPR to edit specific genes, RNA interference to degrade specific mRNA).

Nucleic acid Hybridization

  • Nucleic acid hybridization provides a highly specific way to recognize nucleic acid sequences in vivo and in vitro.
  • FISH: fluorescent in situ hybridization is used to detect and locate specific DNA/RNA sequences.

Alternate Forms of the DNA Double Helix

  • Form depends on both base composition and environment (hydration/salt, protein binding).
    • A, B, and Z forms.
  • All can exist in functional organisms.
  • B form is right-handed; most sequences under physiological conditions take the B form.
  • Z form is left-handed; certain special sequences (in particular, alternating purine-pyrimidine) can take the Z-DNA form (backbone forms a zigzag pattern).
    • Significance: May play a role in transcription and chromosome structure, is recognized by cellular proteins, and may alter chromosome compaction.
  • A form is right-handed; dehydration and protein binding can convert the B form to the A form.

RNA Structure

  • Primary structure of an RNA strand is much like that of a DNA strand, with a couple of exceptions:
    • RNA uses Uracil as a base instead of Thymine.
    • RNA uses Ribose with 2' OH instead of Deoxyribose.
  • RNA strands are typically several hundred to several thousand nucleotides in length.

RNA Secondary and Tertiary Structures

  • RNA is generally single-stranded.
  • RNA molecules can form short double-stranded regions through secondary structure due to complementary base-pairing (A to U and C to G), allowing short regions to form a double helix.
  • Tertiary structure of RNA is determined by:
    • Base-pairing and base stacking within the RNA itself.
    • Interactions with ions, small molecules, and large proteins.