Biochemistry Exam 4 Study Guide

Nucleic Acid Structure

• DNA and RNA Macromolecules
  • Comprised of nucleotides containing a base, sugar, and phosphate group.
  • Bases divided into two groups:
    • Purines: adenine, guanine.
    • Pyrimidines: cytosine, uracil, thymine.
  • Sugars:
    • Ribose: 5 carbons.
    • Deoxyribose: 5 carbons, one less oxygen atom on 2’ carbon vs. ribose.
  • Connections:
    • Bases and sugars: glycosidic bond.
    • Sugar and phosphates: phosphoester bond.
  • Nucleotides linked by phosphodiester linkages.

Structure of DNA Double Helix

• Two helical strands coiled around a central axis forming a double helix.
• Strands are antiparallel (one runs 5’ to 3’, other 3’ to 5’).
• Sugar-phosphate backbone outside, nitrogenous bases inside forming base pairs.
  • AT pairs: 2 hydrogen bonds, GC pairs: 3 hydrogen bonds.
• Dimensions:
  • 10.4 base pairs per turn, length ~34 A, diameter ~20 A.
• Major and minor grooves created by uneven twisting.

DNA Replication

Overview

• Semi-conservative replication: each DNA helix keeps one old and one new strand.

Meselson and Stahl Experiment

• Demonstrated semi-conservative replication using nitrogen isotopes and CsCl centrifugation.

Initiation in Prokaryotes

• Begins at the origin of replication (oriC).
• Involves DnaA, DnaB (helicase), and SSB proteins.

Polymerization in Prokaryotes

• DNA polymerase synthesizes new DNA from 5’ to 3’ using dNTPs.
• Leading strand synthesized continuously; lagging strand in Okazaki fragments.
• DNA Polymerase I and III involved in synthesis and primer removal.
• Topoisomerase enzymes relieve supercoiling.

Prokaryotic Transcription

Overview

• RNA polymerase transcribes DNA to RNA.
• Requires DNA template, ribonucleoside triphosphates.

Initiation

• Involves 35 box, 10 box (Pribnow box), sigma factor binding.

Elongation and Termination

• Elongation builds RNA strand.
• Termination can be rho-dependent or rho-independent.

Lac Operon

• Regulates expression of genes for lactose digestion.
• Controlled by presence of lactose and glucose levels.

Prokaryotic Translation

Genetic Code

• mRNA codons code for amino acids.

tRNA

• Adapter molecule with anticodon and attached amino acid.

Ribosome Structure and Function

• Ribosome reads mRNA, synthesizes protein.
• Consists of large (50S) and small (30S) subunits.
• Three tRNA binding sites: A, P, E.

Polymerase Chain Reaction (PCR)

• Method to amplify DNA sequences.
• Involves cycles of denaturation, annealing, and extension.

Steps

1. Prepare reaction mix with template DNA, primers, etc.
2. Denature DNA at ~95°C.
3. Anneal primers at ~50-65°C.
4. Extend DNA at 72°C using Taq polymerase.

Reverse Transcription PCR (RT-PCR)

• Starts with RNA, converts to cDNA using reverse transcriptase.
• cDNA amplified using PCR.

CRISPR/Cas9

• Gene editing system using guide RNA and Cas9 to cut DNA.
• Used for gene disabling or insertion.

Protein Biochemistry

• Mutagenesis: to study enzyme function.
• Cloning a Gene: involves insertion into vectors for study and manipulation.

Protein Purification Techniques

Cell Disruption

• Methods to lyse cells and release proteins.

Chromatography Techniques

• Ion-Exchange: separates based on charge.
• Affinity Chromatography: uses specific binding interactions.
• HPLC: high-pressure liquid chromatography for separation.

Electrophoresis and SDS PAGE

• Separates proteins by size for analysis.

Assessment of Purity

• Specific activity and SDS PAGE for determining purity of samples.