DNA and RNA - 33

Exam Information

  • Date and Time: Monday, April 21, 5:30-6:20 PM

  • Location: Macbride Hall auditorium

  • Identification: University ID required upon exam submission

  • Calculators: Not allowed on this exam

Lecture Overview

  • Course: BMB 3110

  • Instructor: William Hacker, PhD

  • Topic: Structure of Informational Macromolecules: DNA and RNA

  • Materials: Copyrighted illustrations and texts from Biochemistry, A Short Course, 4th Ed.

  • Outline:

    • DNA and RNA structure

    • Watson-Crick base pairing

    • Meselson-Stahl experiment

    • DNA topology

    • Practice problems: 1-8, 11, 14, 21, 23 in Chapter 33

Central Dogma of Molecular Biology

  • Processes:

    • Replication

    • Transcription (from DNA to RNA)

    • Translation (from RNA to protein)

    • Reverse Transcription (RNA to DNA)

Structure of DNA

  • Composition:

    • DNA is a linear polymer made of monomers:

    • Sugar

    • Phosphate

    • Base

    • The sequence of bases defines the genetic information stored in DNA.

Size and Directionality of DNA

  • Length of DNA molecules:

    • Viruses: polyomavirus (5 Kbp)

    • Bacteria: E. coli (4.6 Mbp)

    • Higher Organisms:

    • Humans (50-300 Mbp/chromosome)

    • Indian muntjac (>1 Gbp/chromosome)

  • Directionality:

    • Chain ends are different:

    • One end has an unlinked 5’ hydroxyl (often phosphorylated)

    • The other end has a free 3’ hydroxyl

    • Sequence notation convention:

    • Written from 5’ to 3’ (e.g., ACG = pApCpG)

Nucleosides and Nucleotides

  • Nucleosides:

    • Composed of a base and a sugar (ribose or deoxyribose)

  • Nucleotides:

    • Include a base, a sugar, and at least one phosphate (can be mono-, di-, or triphosphate, cyclic like cAMP)

    • Example: ATP is adenosine triphosphate

DNA Bases and Deoxyribose

  • Bases attach to the C1’ of deoxyribose:

    • Purines attach at N9

    • Pyrimidines attach at N1

  • The lack of an -OH group at the 2’ position of deoxyribose protects the phosphodiester linkage in the backbone.

Chargaff's Rule

  • States that:

    • %A = %T and %C = %G in a DNA strand

    • This rule predates the determination of the DNA structure.

Structural Features of the DNA Double Helix

  • Two helical strands form a common axis:

    • Right-handed helix

    • Strands are anti-parallel

  • Backbone and bases:

    • Sugar/phosphate backbone on the outside, bases on the inside

    • Bases separated by 3.4 Å, about 10.4 bases per turn

    • Helix diameter: 20 Å

Stabilization of the DNA Helix

  • Stability from:

    • Purine-pyrimidine base pairing

    • Base stacking interactions (individual interactions contribute to cumulative stability)

    • Hydrophobic effect

Major and Minor Grooves of DNA

  • Distinct grooves allow for protein interaction:

    • Major groove (12 Å wide, 8.5 Å deep)

    • Minor groove (6 Å wide, 7.5 Å deep)

  • Allows access to H-bond donors and acceptors for sequence-specific protein interactions.

Alternative Forms of DNA

  • B-form DNA is most common but A- and Z-forms exist:

    • B-form DNA: prevalent in cellular conditions

    • A-form DNA: can form under low humidity, may serve functions in dormant bacteria

    • Z-form DNA: can be induced by negative supercoiling and may play a role in transcription activity

Supercoiling of DNA

  • Can occur when the ends of the duplex are restricted:

    • Can lead to over- or underwinding

  • Two types of variations:

    • Twist: Turns of DNA strands around the helical axis

    • Writhe (supercoiling): Coiling of the helical axis itself

  • Underwound DNA often creates structures called plectonemes.

DNA Compaction and Histones

  • Total DNA in human cells measures around 3.6 m, while nucleus diameter is ~5 µm.

    • Compaction through supercoiling and protein binding

  • Nucleosomes:

    • Core of histone proteins (H2A, H2B, H3, H4) forms a complex with DNA, wrapping about 150 bp of DNA per nucleosome.

    • Interactive roles of histones in DNA packaging and compaction.

RNA vs. DNA

  • Structural Differences:

    • RNA has ribose while DNA has deoxyribose.

    • Uracil replaces thymine found in DNA.

  • RNA can form complex structures, such as stem-loops from complementary base pairing.

Key Concepts to Review for Exam

  • Structure and composition of DNA and RNA

  • Base pairing rules and Chargaff’s relation

  • DNA replication mechanics (including Meselson-Stahl experiment)

  • Differences and similarities between RNA and DNA

  • Understand the roles of histones in DNA compaction and nucleosome formation.

Meselson-Stahl Experiment

  • Concerns DNA replication models: semi-conservative, conservative, dispersive.

  • Method:

    • Grow bacteria in heavy nitrogen (N15), then transfer to standard (N14) media.

    • Use ultracentrifugation to analyze DNA density post-replication.