chap 12 b

CHAPTER 12B: TRANSCIPTION: READING AND EXPRESSING GENES

OVERVIEW OF GENETIC EXPRESSION

  • Gene expression process is primarily catalyzed by transcription and involves the transformation of DNA to RNA.

Key Acronyms and Concepts:

  • RID, ADL, RMED, etc. (Unspecified context in the transcript)

HSP83 RNA TRANSCRIPTION DURING DROSOPHILA EMBRYOGENESIS

  • HSP83 RNA is highlighted in dark blue, indicating its relevance during the development of Drosophila embryos.

REQUIREMENTS FOR IN VIVO TRANSCRIPTION OF EUKARYOTIC CELLS

  1. Cis-acting DNA Elements

    • Promoter

      • Definition: A DNA sequence that initiates transcription.

      • Trans-acting Protein Factor: RNA Polymerase + General Transcription Factors + Mediator.

      • Effect on Transcription: Non-specific; absolutely required for transcription.

    • Enhancer

      • Type of transcription factor: Activator.

      • Effect on Transcription: Specific; induces high levels of transcription.

    • Silencer

      • Type of transcription factor: Repressor.

      • Effect on Transcription: Specific; modulates activator patterns.

    • Insulator

      • Insulator Proteins: Prevent enhancer/silencer from affecting nearby promoters.

MODULAR STRUCTURE OF EUKARYOTIC PROTEIN CODING GENES

  • Cis-regulatory Module (CRM): A collection of transcription factor binding sites.

    • Characteristics:

      • Typically contains enhancer sequences that bind activating transcription factors.

      • Usually found upstream of the promoter & coding sequence, may also be downstream and within introns.

      • Responsible for controlling transcription of adjacent coding regions.

  • Experimentation:

    • Reporter genes are utilized to study the function of CRMs.

    • Cis-regulatory modules are required for cell-specific transcription but are not sufficient on their own; the collection of transcription factors within a cell determines the gene's transcription.

C. ELEGANS EAT-4 TRANSCRIPTION FACTOR BINDING

  • Fig. 12.10 illustrates the binding of EAT-4 transcription factors to three CRMs.

    • Each can bind multiple transcription factors and controls transcription in different subsets of neurons.

    • A close-up of a green CRM indicates it controls transcription in five neurons with unique enhancer sequences.—> Example, will direct the expression of EAT 4 in 5 different neurons of the 38, thereby influencing their functionality and role within the organism.

REAL WORLD RESEARCH EXAMPLE: STICKLEBACK PELVIC FIN EVOLUTION

  • Study Title: The Making of the Fittest: Evolving Switches, Evolving Bodies (HHMI BioInteractive)

  • Research Techniques:

    • Field studies, genome-wide association studies, recombination mapping, DNA sequence analysis, enhancer mapping with reporter genes, gene rescue, paleontology.

TOPOLOGICAL CHALLENGES TO EUKARYOTIC TRANSCRIPTION

  • Chromatin Structure: DNA is associated with proteins.

    • Impact: Chromatin structure prevents transcription factor binding, thereby inhibiting transcription.

Chromatin Structure Dynamics:

  • Heterochromatin:

    • Definition: Closed chromatin that is refractory to transcription factor binding; not transcribed 

  • Euchromatin:

    • Definition: Open chromatin that is available for transcription factor binding and is actively transcribed.

EVOLUTIONARY IMPACTS OF MODULAR GENE STRUCTURE (extra)

  • Mutations from a population are most likely to be eliminated in the coding region

    • Natural selection removes the coding region changes that alter the protein function too much

  • CRM mutations do not change protein function, only the expression pattern

    • CRM changes the expression pattern, not its function, allowing for a greater diversification of traits without adversely affecting the organism's viability.

REAL WORLD EXAMPLES OF CRM CHANGES AND MAJOR CHANGES IN BODY PLAN HOX C6 (extra)

  • Master regulator gene

  • encodes a transcription factor that regulates the expression of genes that determine the thocratic body plan

  • Changes in the Hox c6 CRm change its expression pattern

  • These changes affect the thocratic body plan


GENE EXPRESSION RESULTS FROM:

  • Key Factors:

    • Cis-regulatory module of the gene.

    • Binding of transcription factors.

    • Structure of chromatin surrounding the gene.

EPIGENETICS

  • Definition: The study of heritable changes in phenotype expression that do NOT result from changes in DNA sequence; Responds to our environments and makes changes to our chromatins 

  • Occurrence: Seen in all eukaryotes.

EPIGENETIC MECHANISMS:

  1. Histone Modification—> the addition or removal of chemical groups to histone proteins, which can influence gene expression by altering chromatin structure and accessibility.

  2. DNA Methylation—> the process by which methyl groups are added to the DNA molecule, typically at cytosine bases, leading to gene silencing and affecting overall gene expression patterns.

  3. Non-coding RNAs—> RNA molecules that are not translated into proteins but play crucial roles in regulating gene expression at various levels, including chromatin remodeling, transcription regulation, and post-transcriptional modifications.

HISTONES WITHIN NUCLEOSOMES

  • Histone Tails: Extend beyond nucleosomes and can undergo enzymatic modification.

    • Histone Code: A combination of modifications that affects chromatin openness.

    • Inheritance: Modifications are passed to daughter cells.

  • Example: X-inactivation in mammals, where the histone code contributes to the compaction of the X chromosome into a Barr Body.

EFFECT OF HISTONE ACETYLATION AND METHYLATION ON NUCLEOSOME STRUCTURE

  • Acetylation leads to more open chromatin, while methylation makes it less open.

  • Enzymatic Actions:

    • Histone Acetyltransferase (adds acetyl groups, decreasing tension).

    • Histone Deacetylase (removes acetyl groups, increasing tension).

    • Methylating Enzymes and Demethylases (modify methylation status).

  • Specific Modifications: They are reversible and contribute to the overall histone code determining transcription capability.


More open ←-(Histone acetyltransferase) —> (Histone decetylase) Moderatley open ←-(demethylase) —>(Methaylating enzymes) Less Open 

*add definitions of each transition 

THREE PHASES OF TRANSCRIPTION

  1. Initiation

  2. Elongation

  3. Termination

ELONGATION PHASE

  • Key Elements:

    • RNA Template Strand: This is a single strand of DNA used to synthesize RNA.

    • RNA Transcript: The produced RNA molecule.

    • Coding Strand: Also known as RNA-like strand or sense strand.

    • RNA synthesis begins downstream of the promoter, which is NOT transcribed.

    • Only one DNA strand serves as the RNA template.

    • RNA nucleotides are selected via complementary base pairing with growth in the 5’ to 3’ direction.

PROKARYOTIC TERMINATION MECHANISMS

  1. Rho-dependent termination:

    • Mechanism involves the rut sequence at the 3' end, which is transcribed.

    • Rho protein binds to the rut sequence within the RNA, releasing the RNA from the DNA template.

    • RNA polymerase disassociates from the DNA template.

  2. Rho-independent termination:

    • Involves transcribed termination sequences that induce RNA polymerase release via the formation of a stem-loop structure in the RNA.

Example of Rho-independent Termination Sequence:

  • Transcription of:
    CCACAGCCGCCAGUUCCGCUGGCGGCAUUUUCCACAGCCGCCAGUUCCGCUGGCGGCAUUUU induces RNA polymerase disassociation through stem-loop formation.

EUKARYOTIC TERMINATION OF TRANSCRIPTION

  • It is a less-understood mechanism compared to prokaryotes.

  • Dependent on the type of RNA polymerase involved (e.g., RNA Polymerase II).

  • An integral part of transcript processing.

EUKARYOTIC PRE-mRNA PROCESSING

Pre-mRNA; primary transcript—> both exons and introns are transribed , but introns will be removed during splicing to produce the final mRNA molecule that is ready for translation.

Then:

  1. 5’ Cap added: to the 5' end of the pre-mRNA, which protects the mRNA from degradation and assists in ribosome binding during translation. 

  2. Poly-A tail added: Part of the termination process.

  3. Splicing: Involves the removal of introns.

Addition of 3’ Poly-A Tail & Transcription Termination:

  • Process involves the RNA polymerase transcribing a Poly-A consensus site, with subsequent pausing and cleavage of RNA approximately 30 bases downstream of the consensus site.

  • The RNA polymerase is then released from the DNA template, while poly(A) polymerase adds 100-200 adenine ribonucleotides to the 3’ end of the RNA.

SPLICING

  • Components:

    • 5' Cap

    • Exon 1

    • Intron 1

    • Exon 2

    • Intron 2

    • Exon 3

  • The process of splicing removes introns to produce

    • Final mRNA formed:

    • Exon 1, Exon 2, Exon 3

    • 3' Poly(A)

SPLICESOMES

  • Definition: Cellular machinery responsible for removing introns.

  • Composition: A complex of proteins and small nuclear RNAs (snRNAs).

ALTERNATIVE SPLICING

  • Prevalence: Occurs in most metazoans; approximately 85% of mammalian genes are alternatively spliced.

  • Effect: Increases protein diversity from the 22,000 protein-coding genes in humans which produce over 75,000 proteins.

  • Regulatory Role: Represents an additional layer of gene expression regulation.

NON-CODING RNAs (INCOMPLETE LIST)

  1. Transfer RNA (tRNA): Participates in the translation process.

  2. Ribosomal RNA (rRNA): Serves as a ribosome component, some act as ribozymes during translation.

  3. Small Nuclear RNA (snRNA): Component of the spliceosome.

  4. MicroRNA (miRNA): Essential for eukaryotic gene regulation.

  5. Long Non-coding RNA (Long ncRNA): Role remains unclear, potentially involved in chromatin remodeling.

Genomic Representation:

  • Approx. 2% of the human genome encodes proteins

  • while approximately 65% encodes non-coding RNAs.

MICRO-RNAS (miRNA) AND POST-TRANSCRIPTION REGULATION OF GENE EXPRESSION

  • Overview:

    • ~1700 human genes encode miRNAs.

    • These miRNAs regulate target mRNAs by complementary base pairing.

  • Example Case: Snail Protein critical for dDrosophila embryonic development, with specific miRNAs preventing expression in certain cells through the degradation or translational inhibition of mRNA.

Representation of Regulation:

  • Double-stranded RNA forms between miRNA and target mRNA showing the hybrid interaction.

XIST AS A LONG NON-CODING RNA

  • Function: Required for X-inactivation in mammals. Expressed on inactive X chromosomes only.

  • Mechanism:

    1. Expressed Xist RNA coats the target X chromosome to be inactivated.

    2. Recruits histone-modifying and DNA methylation enzymes.

    3. The X chromosome is compacted into heterochromatin, effectively silencing its expression.

REGULATION OF EUKARYOTIC GENE EXPRESSION

  • Primary Regulation: Occurs primarily at the transcription initiation level.

  • Key Regulation Mechanisms:

    1. Chromatin remodeling (epigenetic changes).

    2. Binding of transcription factors to cis-regulatory modules (enhancers).

    3. Post-transcriptional regulation via splicing and miRNA involvement.