Biotech

Yeast Two Hybrid System

  • Basic concept introduced.
  • Related to proteomics.

Eukaryotic Transcription Regulation

  • In eukaryotes, transcription regulation includes several mechanisms, including transcription factors, enhancers, and insulators.

Enhancer

  • Definition: Enhancers are regulatory DNA sequences that can be far from the promoter but can loop around to interact with the transcription complex.
  • Functionality:
    • Allow for activation of transcription.
    • Can be located up to two million base pairs (2 megabases) away from the gene they regulate.
    • Enhancers interact with the mediator complex, which consists of a variety of proteins.
    • Enhancers enhance transcription by looping to connect with transcription machinery.

Insulator Sequence

  • Definition: Insulator sequences are DNA regions that prevent transcription factors from erroneously activating the wrong genes.
  • Importance: Helps maintain proper gene expression and regulation.

Importance of Gene Expression in Eukaryotes

  • Understanding gene expression is crucial for manipulating eukaryotic genes.
  • Enhancer and insulator sequences can be utilized to control gene expression in different organisms.

Transcription Factors and Dimerization

  • Transcription factors often function as dimers, which interact with each other to regulate transcription effectively.

Epigenetics

  • Definition: Epigenetics refers to changes in gene expression that do not involve alterations in the underlying DNA sequence.
  • Contrast to mutation, which involves changes to the nucleotide sequence.

Mechanisms of Epigenetic Regulation

  1. Histone Post-translational Modifications

    • Histones are proteins that DNA wraps around to form nucleosomes.
    • Type of modifications: Acetylation and methylation.
    • Acetylation: Adds acetyl groups, loosening DNA around histones and enhancing gene accessibility for transcription.
      • Enzyme: Histone acetyltransferase (HAT).
    • Deacetylation: Removes acetyl groups, tightening DNA and inhibiting transcription.
      • Enzyme: Histone deacetylase (HDAC).

  2. DNA Methylation

    • Adding methyl groups to DNA, often resulting in gene silencing.
    • Methylation in eukaryotes silences genes, preventing their expression.
    • Related enzymes include methyltransferases (add methyl groups) and demethylases (remove methyl groups).
    • Methylated sites can recruit HDAC, further silencing transcription.

  3. Nucleosome Remodeling

    • Process of repositioning nucleosomes to allow access to promoter regions for transcription.
    • Involves sliding nucleosomes along the DNA strand to make genes accessible.

  4. RNA-associated Silencing (RNAi)

    • A mechanism involving non-coding RNAs that can silence genes.
    • Example: X-inactivation in females, where one X chromosome is inactivated to balance gene dosage with males.

Heterochromatin and Euchromatin

  • Heterochromatin: Tightly packed, transcriptionally inactive regions of DNA.
  • Euchromatin: Loosely packed, transcriptionally active regions of DNA.

Post-Transcriptional Modifications in Eukaryotes

  • Overview of RNA processing after transcription:
    • Addition of a 5' cap (guanine cap).
    • Addition of a poly-A tail at the 3' end.
    • Removal of introns to form mature mRNA.
  • Mature mRNA exits the nucleus through nuclear pores for translation.

Techniques in Molecular Biology

  • Isolation of mRNA using affinity columns that capture poly-A tails using poly-T oligonucleotides for complementation.

Translation

  • Process of translating mRNA into proteins:
    • Codon recognition, including start codon (AUG) and stop codons (UAA, UAG, UGA).
    • Codons are sequences of three nucleotides that determine the appropriate amino acids during protein synthesis.
    • Anticodons are complementary to codons on tRNA, responsible for bringing specific amino acids to the ribosomes.

Genetic Code

  • Nearly universal, with some exceptions (e.g., UGA coding for tryptophan in microorganisms).
  • Wobble hypothesis allows some flexibility in base-pairing, particularly at the third position of codons, reducing the number of tRNAs needed for protein synthesis.

Importance of Post-Translational Modifications

  • Eukaryotic proteins often require modifications after translation for proper functioning, which prokaryotic systems may not provide.
  • This necessitates choosing suitable host organisms for protein expression, especially if glycosylation or other modifications are needed.

Endosymbiotic Theory

  • Mitochondria and chloroplasts evolved from free-living prokaryotes due to cellular engulfment events.
  • Evidence includes similarities in DNA and ribosomal structure between mitochondria and bacteria (70S ribosomes).

Applications of Epigenetics and Gene Expression Control

  • Understanding transcription regulation allows for genetic manipulation and has implications in biotechnology and health sciences.