chapter 17

Chapter 17: Gene Expression: From Gene to Protein

Overview of Gene Expression

  • Gene expression: The process by which DNA directs the synthesis of proteins.
    • Involves two stages: transcription and translation.
    • Transcription: Creates RNA (specifically, mRNA) from DNA.
    • Translation: Creates proteins from mRNA.
  • Central Dogma of Molecular Biology: \text{DNA} \rightarrow \text{RNA} \rightarrow \text{Protein}
    • DNA is transcribed into RNA, which is then translated into protein.

Beadle and Tatum Experiment

  • Model organism: Neurospora crassa, a type of bread mold that survives on minimal medium (inorganic salts, glucose, and vitamin biotin).
    • Cells were subjected to X-ray radiation to induce mutations.
    • Surviving cells grew into colonies of genetically identical cells.
    • Nutritional mutants: Required supplemented media with amino acids for growth.
    • Established a relationship between genes and enzymes (proteins).
    • Introduced the one gene-one enzyme hypothesis.

Neurospora Experimental Design

  1. Individual Neurospora cells placed in complete medium.
  2. X-rays induced mutations (one gene per cell).
  3. Surviving cells formed genetically identical colonies.
  4. Cells from each colony were placed in minimal medium, with non-growing cells identified as nutritional mutants.
  5. Cells from nutritional mutant colonies further tested with minimal medium plus one additional nutrient to identify specific growth requirements.

Classifications of Mutants

  • Class I mutants: Affect gene A (codes for enzyme A); precursors include ornithine.
  • Class II mutants: Affect gene B (codes for enzyme B); precursors include citrulline.
  • Class III mutants: Affect gene C (codes for enzyme C); precursors include arginine.

Genetic Flow in Cells

  • Transcription Overview:
    • RNA synthesis using DNA information results in mRNA (and other RNA types).
  • Translation Overview: Builds polypeptides based on mRNA information, facilitated by ribosomes.
  • Universality: Transcription and translation are essential in all organisms for metabolic functions.

Key Differences Between Prokaryotes and Eukaryotes

  • Prokaryotic cells:
    • Lack nuclei; DNA, RNA, and ribosomes are in close proximity.
    • Allow for co-transcription translation.
  • Eukaryotic cells:
    • Have nuclei; transcription occurs before translation, with mRNA traveling to cytosol for translation after processing.

Transcription Steps

  1. Initiation: RNA polymerase binds to the promoter region, unwinding DNA.
  2. Elongation: RNA polymerase synthesizes RNA in the 5’ to 3’ direction.
  3. Termination: RNA transcript is released, polymerase detaches from DNA.

Key Features of Eukaryotic Transcription

  • RNA polymerases in eukaryotes: at least three types, with RNA Polymerase II primarily synthesizing mRNA.
  • Eukaryotic promoters include a TATA box for transcription initiation which transcription factors bind to before RNA polymerase II.

RNA Processing in Eukaryotes

  • After initial transcription (producing pre-mRNA), mRNA undergoes:
    1. Addition of a 5’ cap: Modified guanine added to the 5’ end post-20-40 nucleotides transcription.
    2. Addition of a poly-A tail: 50-250 adenine nucleotides added to the 3’ end.
    3. RNA splicing: Removing introns and joining exons to produce the final mRNA product.

Ribozymes and Alternative Splicing

  • Ribozymes: RNA molecules that act as enzymes.
    • Can include intron RNA that catalyzes its own removal.
  • Alternative Splicing: Allows a gene to produce multiple polypeptides depending on included exons.
    • Critical in antibody production for immune diversity.

The Genetic Code Overview

  • Triplet Code: 3 bases correspond to a single amino acid, leading to 64 possible codons from 4 bases.
  • Codon examples:
    • Start Codon: AUG (Methionine).
    • Stop Codons: UGA, UAA, UAG (no amino acid coded).

Amino Acid Sequence Determination

  • Given DNA strands, identify template/non-template, and formulate corresponding mRNA and amino acid sequences.

Reading Frames and Mutation Implications

  • Reading frames: Codons are read in groups of three; shifting this frame leads to a different amino acid sequence and potential mutations.
  • Mutations:
    • Changes in genetic information can occur spontaneously or via external influences (mutagens).
    • Point mutations: Change in single nucleotide pairs, may lead to different resultant polypeptides.
    • Insertions/Deletions: Can cause significant downstream effects by shifting reading frames.

Types of Mutations

  1. Nucleotide-Pair Substitution:
    • Silent mutation: No change in amino acid.
    • Missense mutation: Change results in a different amino acid.
    • Nonsense mutation: Produces a stop codon.
  2. Insertions and Deletions:
    • Cause frameshifts if not in multiples of 3.

Gene Editing and CRISPR-Cas9

  • Gene editing allows precise gene alteration, enhancing capabilities for medicinal applications including genetic disease treatments. Recent advancements involve the CRISPR-Cas9 system.