Operon Structure and Function

Operon Structure and Function

• Operons are gene expression control units in bacteria.
• They consist of:
  • Controlled genes
  • Promoter region (RNA Polymerase binding site)
  • Operator region (on/off switch)
• Repressible operons (e.g., tryptophan):
  • Usually "on," but can be turned "off."
  • A repressor protein is inactive until activated by a corepressor (e.g., tryptophan).
  • Binding to the operator prevents RNA polymerase from transcribing genes
• Inducible operons (e.g., lactose):
  • Usually "off," but can be turned "on."
  • An active repressor protein is bound to the operator.
  • An inducer (like lactose) binds to the repressor, inactivating it, and allowing transcription.

Operon Components

• Promoter: Binding site for RNA polymerase.
• Operator: Controls gene expression. Can block transcription if bound to a repressor.
• TATA box: Within promoter, signals RNA polymerase binding location
• Genes: Code for proteins.
• Terminator: Stops transcription
• These key components are essential for bacterial gene regulation, in particular responding to environmental conditions.

Bacterial Gene Regulation

• Gene expression regulation often involves feedback loops, where regulating protein or molecule levels adjust an enzyme's activity.
• The cell can stop making enzymes if regulated molecules become too abundant by blocking transcription of genes.

Regulatory Genes

• Regulatory genes produce repressor proteins.
• Repressor proteins block gene expression by binding to operators.
• Signals (like lactose) cause repressors to detach, allowing transcription.
• This saves energy by turning genes off when not needed.

Eukaryotic Gene Components

• Coding region: Contains protein-coding instructions (exons and introns).
• Regulatory regions: Control gene activity.
  • Promoter: RNA polymerase binding site.
  • Enhancers/Silencers: Increase/decrease gene expression .
• Non-coding regions: 5' and 3' untranslated regions, introns, and polyadenylation signal (important for mRNA processing and stability).

Chromatin Structure and Gene Regulation

• Euchromatin: Loosely packed chromatin; DNA is accessible for transcription.
• Heterochromatin: Tightly packed chromatin; DNA is inaccessible; transcription is suppressed.
• DNA methylation or histone acetylation can alter chromatin structure and thus gene expression.

DNA Methylation and Histone Acetylation

• DNA methylation: Adding methyl groups to DNA; typically correlates with gene silencing.
• Histone acetylation: Adding acetyl groups to histone proteins; typically correlates with gene activation by loosening their grip.

Epigenetic Inheritance

• Epigenetic inheritance is the transmission of gene expression changes to offspring without changing the DNA sequence.
• Modifications (e.g., methylation, acetylation) are passed on.

Noncoding RNA (ncRNA)

• ncRNA regulates gene expression without coding for proteins.
• Examples include small nuclear RNAs (snRNAs).

Virus Structure and Replication

• Viruses are non-cellular infectious agents, consisting of genetic material (DNA or RNA) inside a protein coat (capsid). Some have an outer envelope.
• They replicate only within host cells, hijacking the host's machinery.

Lytic and Lysogenic Cycles

• Lytic cycle: Viruses replicate immediately, destroying the host cell.
• Lysogenic cycle: Viral DNA integrates into the host genome; replication occurs without immediate host destruction.

Retroviruses

• Retroviruses have RNA genomes.
• Reverse transcriptase converts viral RNA into DNA, which integrates into the host's DNA.

Viruses, Prions, and Viroids

• Viruses: Genetic material (DNA or RNA) inside a protein coat.
• Prions: Misfolded proteins; cause other proteins to misfold, causing degenerative diseases.
• Viroids: Small circular RNA molecules infecting plants; lack protein-coding ability.

Nucleic Acid Hybridization

• Nucleic acid hybridization techniques are used to detect specific DNA or RNA sequences in complex samples. This is more efficient than prior methods.
• It permits quick identification of gene sequences in complicated samples.

DNA Sequencing (Dideoxy Chain Termination)

• DNA sequencing uses dideoxynucleotides to terminate DNA chain extension at specific points, generating fragments that differ in length.
• Fragment lengths are determined to identify the sequence.

Enzymes in Biotech

Enzymes are used to cut, copy, and otherwise manipulate DNA and RNA within biochemical applications.

Nucleic Acid Probes

• Nucleic acid probes are labeled DNA or RNA segments used to locate specific sequences in a sample.
• They are used in hybridization to identify expressed genes by binding to their mRNA counterparts.

Restriction Enzymes

• Restriction enzymes cut DNA at specific sequences.
• In bacteria, these enzymes protect against viral DNA.
• Different restriction enzymes produce blunt or sticky ends.
• Sticky ends facilitate the creation of recombinant DNA.

Cloning a Eukaryotic Gene

The plasmid vector is cut with a restriction enzyme and the eukaryotic gene is inserted into the cut plasmid using DNA ligase. The recombinant plasmid is inserted into a host bacterium to produce copies of the gene.

cDNA Production

• cDNA (complementary DNA) is generated by reverse transcribing mRNA. This avoids the need to consider introns. -mRNA is used as a template.

Yeast vs. Bacteria as Cloning Hosts

• Yeast is generally a better host for eukaryotic cloning because of their ability to support complex processes.
• Yeast can easily support plasmids and also are conducive to manipulation.

PCR (Polymerase Chain Reaction)

• PCR is a technique used to exponentially amplify a specific DNA sequence.
• Repeated cycles of heating and cooling are used to produce many copies.

Gel Electrophoresis

• This technique separates molecules (DNA, RNA, proteins) based on size, charge, and shape.

Restriction Fragment Length Polymorphism (RFLP)

• RFLP analysis detects differences in DNA fragment lengths due to variations in restriction enzyme cutting sites.

Stem Cells (Totipotent vs. Pluripotent)

• Totipotent: Early embryonic stem cells can differentiate into any cell type.
• Pluripotent: Late embryonic stem cells can differentiate into multiple cell types.

Human Genome Project

• Led to the complete sequencing of the human genome.
• Revealed structural details, genetic diversity, disease genes, and gene regulation.

SNPs (Single Nucleotide Polymorphisms)

• SNPs are small genetic variations; used as markers and to study disease susceptibility and drug response.

Chromosomal Errors (Duplication, Translocation, etc.)

• Errors can lead to new gene combinations, which can be useful for evolution.

Homeobox/Hox Genes

• Homeobox genes control embryonic development, especially body plan formation and the structure of the body segments.