Chapter 14: Gene Regulation in Bacteria

• Module 8 focuses on gene regulation in bacteria and introduces eukaryotic gene regulation.
• Progress report 3 on Safety and Ethics is due at the end of the week.

Chapter 14: Gene Regulation in Bacteria

• Gene regulation involves varying gene expression based on conditions.
• Constitutive genes are always expressed (unregulated).
• Gene regulation is crucial for metabolism, environmental stress response, and cell division.
• Gene regulation occurs by controlling transcription rate via regulatory proteins.
• Translational repressors bind to mRNA.
• Feedback inhibition: the pathway's product inhibits the first enzyme in the pathway.
• Regulatory proteins and small effector molecules play key roles.
• Regulatory proteins:
  • Repressors bind to DNA and inhibit transcription.
  • Activators bind to DNA and increase transcription.
• Small effector molecules affect transcriptional regulation.
• Inducers bind to activators/repressors, allowing regulatory transcription factors to bind to DNA to either promote or inhibit transcription.
  • Bind to activators and repressors
• Inhibitors:
  • Corepressors bind to repressors and bind them to DNA
    • bind to repressors and bind them to DNA
  • Inhibitors bind to activators and prevent binding
• The lac operon's organization optimizes bacterial lactose utilization
  • Regulatory unit consisting of a promoter that condrtols coding genes which encode polycistronic mRNA
    • Components:
      • Promoter: bonds to RNA polymerase
      • Operator: binds to lac repressor protein
      • CAP site: Binds to the Catabolite Activator Protein
      • Terminator: Ends transcription
      • Coding genes: lac Z, lacy, lacA
• Repressor protein regulates transcription of the lac operon through the repressor protein and an activator protein.
  • First is inducible negative control mechanism involving lac repressor
    • Allolactose binds to lac repressor and prevents the repressor from binding to the DNA
• The repressor protein binds tightly to the operator site and inhibits the ability of RNA polymerase when no allolactose is present
• When present allolactose binds to repressor, altering conformation and preventing binding, allow RNA pol to work
• Steps for repression/activation:
  • Lactose present: converts to allolactose and binds to repressor
  • Lac protein synthesized: RNA polymerase can transcribe lac operon protein so lactose uptake efficient'
  • Lactose is depleted: allolactase levels decrease and detached from repressir
  • Lac proteins degrade: proteins get used up or degrade
• Experiment 14A involved introducing lac operon portions into different strains and labeled plasmids with portions of operon as F'.
  • Hypothesized that lacl- mutation results in synthesis of an internal inducer
• If correct: inducer protein produced from the chromosome can diffuse and activate the lac operon on the F' factor
• Alternative: lacl- mutation eliminates function of lac repressor that can diffuse throughout cell
  • If correct the repressor on F' factor can diffuse and turn off lac operon on bacterial chromosome
• Results:
  • Mutant strain → no addition of lactose → 100% B galactosidase
  • Mutant strain → addition of lactose → 100% B-galactosidase
  • Merzygote → no addition of lactose → < 1%
  • Merzygote → addition of lactose → 220% production
• Explanation
  • Repressor encoded on the F' factor represses both operons in absence of lactose
  • Trans effect: genetic regulation occur even though DNA segments are not adjacent
    • Complement dby introduction of second gene with normal function
  • Cis-effect: DNA sequence must be adjacent and mediated by sequences that bind RTFs
    • No affected by introduction of normal element
• A lacI mutation prevents repressor production, leading to constitutive expression of the lac operon and is rescued by a functional lacl in a merodiploid since the repressor acts in trans.
• A lacOc mutation alters the operator so the repressor cannot bind, also causing constitutive expression. However, it cannot be rescued by a wild-type laco in a merodiploid since the operator acts in cis.
• Both lacl and lacOc mutations lead to continuous lac operon activation, but lacl is trans-acting and rescuable, while lacOc is cis-acting and not rescuable.
• Interpreting Diauxic growth:
  • Sequential use of two sugars by a bacterium is diauxic growth
    • Graph begins positively as glucose is used and number of bacteria increase and then there is a flat lag phase before proceeding with different sugar
• cAMP (cyclic AMP) - CAP complex is an example of trancriptional regulation that is inducible under positive control
  • Complex binds to CAP site near lac promoter and increase transcription
  • Glucose inhibits adenyl cyclase which decreases cAMP levels
  • Lactose promotes formation of complex and increases transcription
• Lactose and Glucose = low cAMP
• lac operon contains three operator sites:
  • 01 → next to promoter
  • O2 → downstream in lacZ region
  • O3 → slightly upstream of promoter
  • 2 of 3 required for repressor to repress transcription
  • Mutations showed that transcription repression can't occur without a combination of two operator sites present
• The Lac repressor (Lacl) binds to the primary operator (01) and one of the auxiliary operators (O2 or O3), forming a DNA loop which enhances repression by stabilizing Lacl binding and reducing RNA polymerase access to the promoter.
• Riboswitches regulate gene expression by binding small molecules to alter RNA structure, affecting transcription or translation differently:
  • Controls formation of a transcription terminator (Transcription)
  • Controls accessibility of the ribosome-binding site (RBS) (Translation)
• ON vs. OFF
  • Ligand binding induces a terminator hairpin, causing RNA polymerase to dissociate (OFF) (Transcription)
  • Ligand binding causes an RNA structure that blocks the RBS, preventing ribosome binding (OFF) (Translation)
• Key Structural Elements
  • Antiterminator vs. terminator stem-loops
  • Sequestered vs. exposed RBS
• Riboswitches rely on alternative RNA conformations but act at different stages of gene expression.

Chapter 15: Gene Regulation in Eukaryotes I: Transcriptional Regulation

• Gene Regulation: level of gene expression is varied

  • Produces different cells types in multicellular species
  • Facilitate changes during development
  • Respond to environmental changes such as nutrient availability

• Occurs during...

  • Transcription: regulatory transcription factors, arrangement and composition of nulceosomes, and DNA methylation
  • RNA Modification: alternative splicing and RNA editing
  • Translation: proteins regulate translation or mRNA degradation, and RNA interference
  • Posttranslation: feedback inhibition and covalent modifications regulate protein function

• Combination Control: results from the many factors that contribute to gene regulation

  • One or more activator proteins
  • One or more repressor proteins
  • Activators and repressors may be modulated bu binding of small effector molecules
  • Regulatory proteins may alter nucleosomes near promoter
  • DNA methylation may inhibit transcription

• Transcription Factors: proteins that influence ability of RNA polymerase to transcribe a given gene

  • General transcription factors:
    • Required for binding of RNA pol to the core promoter and its progression
    • Necessary for basal transcription
  • Regulatory transcription factors
    • Serve to regulate the rate of transcription of target genes
    • Influence ability of RNA polymerase to begin transcription of a particular gene

• Regulatory trancription factors recognize regulatory elements

  • Sequences are known as regulatory elements, control elements, or regulatory sequences
  • Enhancer: DNA region that contains one or more regulatory elements
  • Actovators are regulatory proteins that increase transcription while repressors do the opposite

• Three Ways to Modulate Regulatory Transcription Factors:

  • Binding of small effetor molecule such as hormone
  • Protein protein interactions (between RTFs) cause binding to enhancer
  • Covalent modifications such as phosphorylation

• Chromatin Remodeling Enzymes: Structure

  • -Function:
    • Alter positions and compositions of nucleosomes
    • Position of nucleosomes change
    • Eviction of histone octamers occurs
    • chang e in nucleosome composition
  • Closed conformation: tightly packed and transcription is difficult
  • Open conformation: chromatin accessible to transcription factors

• Histone Variants:

  • Defnition: histone genes that have accumulated mutations
  • Incorporated into subset of nucleosomes to create specialized chromatin
  • Some variants promote open chromatin, and some promote closed which alters gene transcription

• Histone Code:

  • Definition: the pattern of modifications provide binding sites for proteins that promote alterations in chromatin structure
  • Proteins bind to histones based on the code and affect transcription
  • Histone modifications include
    • Methylation
    • Acetylation: can aliminate positive charge in lysine and decrease binding
    • phosphorylation

• DNA methylation and Gene Expression:

  • Definiton: covalent attachment of methyl groups and carried out by DNA methyltransferase
  • DNA methylation usually inhibits eukaryotic gene transcription

• CPG islands:

  • Contain high number of CpG sites and are unmethlyated in housekeeping genes
  • In tissue specific genes: the expression of genes silineced by CpG methylation which affects binding of transcription
  • methyl-CpG-binding protein recruits other proteins that change the chromatin to closed conformation

• Hertidity of Methylation:

  • Inherited during cell division
  • Specific genes are methylated in gamests from female or male parent via de novo methylation -Pattern of one copy of the gene being methylated and the other not is maintained in the resulting offspring via maintenance methylation

• Nucleosome free region:

  • Beginning and end of many genes
  • Nucleosomes tend to be precisely positioned near the beginning and end of a gene, but are less regularly distributed elsewhere

• Preinitiation complex:

  • Binding of activators: activation protein binds to regulatory elements within enhancer sequences. The enhancer may be close to transcriptional start site or far away
  • Recruitment of coactivators: activators bind directly to the DNA of regulatory elements within the enhancer, then recruit coactivators
  • Chromatin remodeling and histone modification: an activator protein recruits a chromatin remodeling complex and a histone mdoification enzyme
  • Formation of preinitiation compelx: general transcription factors and RNA polymerase II are able to bind to the core promoter and forma preinnitation complex

• Elongation phase of Transcription in Eukaryotes:

  • Formation of an open complex: DNA strand must be separated into an open complex so that one of them can act as a template for RNA synthesis
  • Promoter escape: at core promoter GTFs and mediator bind to RNA polymerase II and prevent it from taveling along the template strand. For elongation to occur RNA polymerase must be released from this binding
  • Proximal promoter pausing involves:
    • RNA polymerase II pauses in RNA synthesis while close to transcription start site
    • Involves binding of two factors, DSIF and NELF
    • To release pause, positive trancriptional elongation factor b phophorylates both DSIF and NELF, which results in release of NELF and causes DSIF to facilitate elongation
  • Histone modifications: Histone-modifying enzymes play akey role in histone removal and placement during the elongation phase of transcription, through histone acetylation, H3 methylation, and H2B ubiquitination

• The glucocorticoid receptor (GR) is activated by binding glucocorticoids, leading to its translocation into the nucleus, where it regulates gene expression by binding glucocorticoid response elements (GREs) or interacting with transcription factors like NF-κB to modulate inflammation, metabolism, and stress responses.

• CREB (cAMP response element-binding protein) is activated by phosphorylation via PKA, CaMK, or MAPK in response to cAMP or other signals. It binds cAMP response elements (CREs) in DNA, recruiting CBP/p300 to enhance transcription of genes involved in metabolism, neuronal plasticity, and survival.

• ChIP-Seq (Chromatin Immunoprecipitation Sequencing) is a technique used to analyze protein-DNA interactions genome-wide by crosslinking proteins to DNA, shearing the chromatin, immunoprecipitating with an antibody specific to a DNA-binding protein (e.g., transcription factors, histones), purifying the bound DNA, and sequencing it. This identifies binding sites and regulatory elements, helping to study gene regulation, epigenetic modifications, and transcription factor activity.