Lecture 23_24

Regulation of Gene Expression in Eukaryotes

Goals

  • Understand how gene expression is controlled in eukaryotes through various mechanisms of regulation.

  • Review previous concepts related to transcription regulation.

Regulation of Transcription (Eukaryotes)

  • Key Questions:

    • WHERE does transcription take place?

    • WHEN is transcription initiated?

    • WHY is transcription regulated?

    • HOW does transcription regulation occur?

Control of the Lac Operon

  1. In the Presence of Glucose (Negative Control):

    • Transcription of the lac operon is blocked.

  2. In the Absence of Glucose and the Presence of Lactose (Induction):

    • Transcription of the lac operon occurs.

  3. Positive Control of Lac Operon:

    • Involves cAMP and CAP.

Components of the Lac Operon

  • lacl Gene

    • Produces the repressor protein that is constitutively expressed.

    • Wild Type: 1+ O+ Z+ Y+ A+ (normal operon) — repressed when no lactose is present.

    • Mechanism:

    • The repressor binds to the operator region of the lac operon, blocking transcription.

    • Result: No transcription; no enzymes are produced.

Induction Mechanism (Absence of Glucose and Presence of Lactose)

  • Wild Type Configuration:

    • 1+ O+ Z+ Y+ A+ (lactose present).

    • When lactose is present, it binds to the repressor, altering its conformation, thus preventing binding to the operator.

    • Outcome:

    • Transcription and consequently translation occur, resulting in the production of enzymes required for lactose metabolism.

Inversely Related Components of Transcription

  • The concentration of cAMP is inversely related to the concentration of glucose.

  • High Glucose Conditions:

    • Adenylyl cyclase is INACTIVE, leading to LOW cAMP levels.

    • As a result, CAP (Catabolite Activator Protein) does not bind to DNA, causing infrequent transcription of the lac operon as the cell continues using glucose as its primary energy source.

  • Low Glucose Conditions:

    • Adenylyl cyclase is ACTIVE, leading to HIGH cAMP levels.

    • CAP-cAMP complex binds to DNA, promoting frequent transcription of the lac operon to utilize lactose as an energy source when glucose is scarce.

Overview of Gene Expression Regulation in Eukaryotes

  1. Regulation of Chromatin Structure

  2. Regulation of Transcription

    • Formation of pre-mRNA (primary transcript).

  3. Regulation of Splicing and Processing

    • Converting pre-mRNA to mature mRNA.

  4. Regulation of Transport

    • mRNA transit through nuclear pore to cytoplasm.

  5. Degradation of mRNA

  6. Translational Regulation

  7. Post-Translational Modifications

Chromatin Structure

  • Components of Chromatin Regulation:

    • Chromatin is organized in a manner that influences gene expression.

      • Gene-poor chromosomes occupy less active territory within the nucleus, while gene-rich chromosomes are located in areas conducive to transcription.

  • Alteration of Chromatin Structure:

    • Open Configuration:

      • Histone Acetyl Transferases (HATs) add acetyl groups to lysines, enhancing transcription by promoting a looser chromatin structure.

    • Closed Configuration:

      • Histone Deacetylases (HDACs) remove acetyl groups, condensing chromatin and inhibiting transcription.

Histone Modifications and Their Effects

  • Positively Charged Tails of Nucleosomal Histones:

    • Interaction with negatively charged phosphate groups of DNA.

    • Acetylation:

      • Weakens the interaction between histones and DNA, thereby facilitating access for transcription factors and RNA polymerase.

Role of Chromatin Remodeling Complexes

  • Chromatin remodeling alters DNA-protein contacts, changes DNA paths, and repositions nucleosomes to facilitate transcription.

DNA Methylation and Gene Expression

  1. The degree of DNA methylation is inversely related to gene expression, where higher methylation correlates with lower transcriptional activity.

  2. Methylation patterns are tissue-specific and can be inherited by daughter cells.

  3. Determining Methylation State:

    • Conducting restriction enzyme analysis using enzymes like HpaI and MspI to distinguish between methylated and non-methylated DNA regions.

Regulatory Promoters and Enhancers

  • Regulatory Promoter Elements:

    • Core promoter contains essential sequences (TATA box, CAAT box, GC box) essential for transcription initiation.

    • Enhancers are modular sequences that interact with transcription factors to boost expression, whereas insulators prevent enhancers from affecting inappropriate genes.

Example of Transcriptional Control

  • In response to galactose, GAL4 activates transcription:

    • In absence of galactose, GAL80 blocks GAL4.

    • Upon galactose binding to GAL3, GAL80's conformation changes and allows GAL4 to activate transcription.

Splicing and Processing of mRNA

  1. Initial pre-mRNA transcript gets modifications such as:

    • Addition of 5' cap.

    • Creation of Poly-A tail.

    • Removal of introns through splicing.

  2. Alternative Splicing:

    • Enables generation of multiple proteins from a single gene.

    • Example: calcitonin vs CGRP peptide generation from the same mRNA.

miRNA and Gene Silencing

  • Mechanism of Action:

    • Encoded by miRNA genes, these regulate gene expression by degrading target mRNAs or inhibiting their translation, mainly by binding to the 3' untranslated region (UTR).

    • RNA Interference (RNAi):

      • Generated by Dicer which cleaves dsRNA into siRNAs.

      • siRNAs direct RISC to degrade complementary mRNA or inhibit translation if there are mismatches.

Questions on Regulation

  • Which two mRNA structures interact for stability?:
    a. Adjacent introns and exons
    b. 5’ cap and first exon
    c. 5’ cap and 3’ poly(A) tail
    d. All of the above

  • Which does NOT inhibit gene expression?
    a. Cleaving mRNA
    b. Altering chromatin structure
    c. Stimulating RNA degradation
    d. Stabilizing translation machinery

Transcription Regulatory Networks

  • Transcription factors and target genes form directed graphs representing complex regulatory networks.

  • The regulatory interactions exhibit scale-free topology indicative of regulatory hubs with local motifs and modules.

  • Despite similarities across organisms, variations exist in the qualitative differences among components.

References:

  • Each concept is taken from various academic sources including "Genetics: A Conceptual Approach" and current scientific literature on gene expression regulation in eukaryotes.

  • Significant insights based on endomesoderm specification models have been provided by various research labs, illustrating updated regulatory interactions pertinent to characterization of gene expression dynamics.