🧬

Biochemistry Exam Study Guide

May 3, 2025

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.