Regulation of Transcription

Transcription is the process by which DNA is used as a template to make mRNA via an enzyme called RNA polymerase. Transcription is an important step in gene expression, and as such, it is highly regulated. In prokaryotes, genes are grouped together into DNA sequences, known as operons, that can be induced or repressed to regulate expression of these genes together. Regulation in eukaryotes is much more complicated and involves a number of transcription factors and regulatory sequences of DNA. Epigenetic mechanisms, including how the DNA is packaged, also play a role in transcription regulation by controlling what segments of DNA are available to the RNA polymerase.

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Overview

Review of transcription

Definition:

  • Central dogma: To express a gene, DNA is transcribed into RNA, which is then translated into a protein (or a protein fragment known as a polypeptide).
  • Transcription is the process by which DNA is used as a template to make mRNA.
  • RNA polymerase: enzyme that “reads” the DNA template strand and creates the mRNA
  • Promoter sequence: sequences of DNA just upstream from the target gene that indicate the start site and direction of the gene

DNA:

DNA is a double-helix molecule made up of 2 antiparallel strands. DNA has a structure that looks like a twisted ladder.

  • The “sides” of each ladder are made up of alternating deoxyribose (a 5-carbon sugar) and phosphate molecules.
  • The “rungs” of the ladder are made up of matched nitrogen-containing molecules called nucleotides, frequently referred to as “bases.”
  • DNA base pairs:
    • Guanine (G), cytosine (C), adenine (A), and thymine (T)
    • G pairs with C (and vice versa) via 3 hydrogen bonds.
    • A pairs with T (and vice versa) via 2 hydrogen bonds.
    • These base pairs can be “read” as a string of letters(e.g., GTATCGA).
    • This string of letters is the “code,” or instruction manual, that is ultimately used to create proteins.
  • Grooves:
    • The DNA helix is asymmetrical as it rotates.
    • This rotation creates major and minor grooves between coils. 
    • The major groove is wide enough that many regulatory proteins can bind directly to the DNA through this space.

RNA:

  • A single-stranded molecule made up of alternating ribose (a 5-carbon sugar) and phosphate molecules
  • Each ribose is bound to an RNA nucleotide:
    • Guanine (G), cytosine (C), adenine (A), and uracil (U)
    • Note that instead of thymine, A binds with U (and vice versa) via 2 hydrogen bonds.

Structure of RNA and DNA

Image by Lecturio.

Overview of transcription regulation

  • Many different types of signals can influence whether or not a particular gene is transcribed. Examples of these signals include:
    • Hormones
    • Enzymes
    • Pharmaceutical agents
    • The presence or absence of certain nutrients or other molecules (e.g., lactose or tryptophan)
  • Mechanisms of transcription regulation:
    • Controlling access of polymerase to the target DNA sequence via:
      • Transcriptions factors
      • Enhancers and repressors
      • DNA packaging
    • Controlling elongation of the RNA via elongation factors/activators
    • Controlling termination of the polymerase
  • Positive regulation: 
    • Gene expression is increased.
    • Uses positive regulators, activators, or enhancers
  • Negative regulation:
    • Gene expression is decreased.
    • Uses negative regulators, repressors, or insulators.

Regulatory Proteins

Transcription regulation is mediated in part by regulatory proteins that can bind to the DNA in its helical form (unwinding is not necessary) and regulate the transcriptional activity of RNA polymerase.

Overview of regulatory proteins

  • Regulate transcription by binding to regulatory sequences of the DNA:
    • Promoters
    • Enhancer sequences
    • Insulator sequences 
  • Regulatory proteins have different domains:
    • DNA binding domain: portion of the protein that binds to the DNA
    • Functional domain: portion of the protein that interacts with the DNA and/or other proteins to carry out its function
  • Regulatory proteins typically contain 1 of 3 primary DNA binding motifs:
    • Helix–turn–helix
    • Zinc fingers
    • Leucine zippers

Helix–turn–helix

Regulatory proteins with the helix–turn–helix DNA binding motif have the following characteristics:

  • Structure: 2 helical segments oriented perpendicular to one another and connected to each other via a looping segment of protein
  • Looping segment contains the functional domain
  • Proteins bind the DNA through the major groove
  • Often work in pairs
  • A subset of helix–turn–helix proteins are known as homeodomain proteins, which are regulatory proteins often involved in development.

2 helix–turn–helix regulatory proteins bound to DNA in the major groove

Image by Lecturio.

Zinc finger

  • 3 finger-like structures interact with DNA through the major groove.
  • Uses zinc to closely associate with the DNA
  • This same motif is seen in steroid receptors, which contain a zinc-finger domain → allows the activated receptors to bind directly to the DNA and directly affect transcription

The zinc-finger binding motif:
Often used in steroid receptor binding mechanisms

Image by Lecturio.

Leucine zipper

Leucine zippers consist of 2 proteins, each containing a helical subunit and a hydrophobic subunit.

  • The helical subunits:
    • Enter the DNA through the major groove
    • Associate with opposite ends of the DNA within the groove
  • The hydrophobic subunits:
    • Remain outside the DNA (because DNA is hydrophilic)
    • Contain leucine molecules that “zip” the two proteins together
  • Additional portions of the proteins contain functional domains that can then interact with the DNA or surrounding proteins.

The leucine-zipper binding motif:
Has 2 subunits that zip together

Image by Lecturio.

Prokaryotic Transcription Regulation

Overview

  • Prokaryotic gene expression is regulated primarily at the level of transcription.
  • Prokaryotic genes are organized into groups called operons.
  • Transcription regulation is primarily through inducing or repressing the operons.
  • Genes are always on in the absence of other factors (e.g., repressors) 

Operons

Operons are clusters of coregulated genes plus all the components that come together to regulate those genes. Operons contain 2 primary regions of DNA sequences:

  • Regulatory region contains:
    • Promoter: binds sigma factor and RNA polymerase to initiate transcription
    • Operator: a DNA sequence that can bind a repressor 
      • Repressor: proteins that prevent RNA polymerase from moving down the gene 
      • Transcription is inhibited when repressors bind to their operators.
    • Regulatory gene: genes for an activator or repressor protein
    • Promoter for the regulatory gene
  • Coding region: 
    • Often contains multiple genes for several different proteins
    • These genes are either all turned on or all turned off.

Example of a prokaryotic operon:
Prokaryotic gene expression is regulated primarily at the level of transcription.
Operons are clusters of coregulated genes in prokaryotes.

Image by Lecturio.

Inducible operons

Inducible operons are operons that are off in the “normal” state and turn on under certain conditions. A common example is the lac operon in Escherichia coli. Under normal circumstances, E. coli uses glucose for energy. When glucose is unavailable and/or lactose is present, the presence of lactose induces the lac operon:

  • Coding region: contains genes involved in lactose metabolism
  • Regulatory region codes for:
    • A repressor
    • An activator: catabolite activator protein (CAP)

Lac operon:
In the presence of lactose, expression is activated.

Image by Lecturio.

Gene regulation via the lac repressor:

  • Lactose prevents the binding of the repressor to the operator sequence
  • In the presence of lactose: repressor is unable to bind → transcription of the genes can occur
  • When lactose is absent: repressor binds to the operator sequence → transcription is inhibited because the RNA polymerase is blocked
  • Therefore, the presence of lactose induces the transcription of genes for lactose metabolism.

Gene regulation via the CAP activator:

  • When glucose is low: CAP is produced → binds to the enhancer region (see “Enhancer sequences” below) → activates/accelerates transcription
  • When glucose is abundant: CAP is not produced → transcription is not enhanced

Repressible operons

Repressible operons are operons that are on in the “normal” state and turn off under certain conditions. The trp operon is a common example:

  • Coding region contains several genes that code for enzymes necessary for tryptophan synthesis.
  • :Regulatory region codes for a repressor
    • The repressor requires tryptophan in order to be activated.
    • Without tryptophan, the repressor is inactive.

The default state is for the operon to be on and producing tryptophan.

  • When tryptophan is present:
    • Tryptophan binds to its repressor → repressor is activated
    • The repressor binds to its operator → prevents transcription
    • Result: The presence of tryptophan turns off the genes that would make more tryptophan.
  • When tryptophan is not present:
    • There is no tryptophan to bind to the repressor → the repressor is inactive
    • Repressor cannot bind to the operator → transcription occurs
    • Result: When no tryptophan is present, the genes that code for enzymes to synthesize tryptophan are transcribed.

Trp operon:
Tryptophan activates the repressor.

Image by Lecturio.

Eukaryotic Transcriptional Regulation

Overview

  • Regulation of transcription in eukaryotes is more complex than in prokaryotes because the development of eukaryotes is vastly more complicated. 
  • Eukaryotic genes are always off in the absence of a multitude of transcription factors that must be recruited. 
  • Eukaryotic gene expression is regulated at every step of the pathway:
    • DNA packaging in chromatin
    • Transcription
    • Posttranscriptional modification
    • Translation
    • Protein modification and degradation
  • Transcription is regulated primarily by:
    • Transcription factors
    • Regulatory sequences of DNA

Transcription factors

Transcription factor (TF) is a generic term for proteins necessary for transcription. Each of these factors helps to regulate gene expression.

  • General transcription factors:
    • Bind to the promoter sequence
    • Are required for the binding of RNA polymerase II to the DNA to initiate transcription
  • Initiation complex: 
    • The complex of transcription factors and RNA polymerase II at the promoter sequence
    • Once the initiation complex is assembled on the promoter, transcription can begin.
  • Transcription-associated factors: specific proteins that act in a time- or tissue-dependent manner to initiate transcription 

Enhancer sequences

Enhancer sequences of DNA help to initiate or augment transcription, which further promotes gene expression.

  • These sequences can bind to:
    • Activator proteins: 
      • Specific types of TFs that help to assemble and/or interact with TFs on the promoter 
      • Function to activate or augment transcription
    • Repressor proteins: repress transcription
  • Usually located several thousand base pairs upstream of the target gene
    • Create a loop in the DNA when they interact with the promoter
    • Allow for further fine-tuning of regulation:
      • A single enhancer sequence may interact with multiple promoters/genes.
      • A single gene may interact with multiple enhancer sequences.
  • Insulator sequences: other sequences of DNA that can prevent the enhancers from looping to interact with the promoter region

Epigenetics

Epigenetic regulation is the regulation of gene expression that does not involve alterations to the DNA sequence or any of its transcribed products. Epigenetics include:

  • How accessible the DNA is to RNA polymerase based on how it is packaged: DNA is wrapped around a nucleosome with a histone tail.
  • Histone modification via:
    • Acetylation → “unpacks” DNA and allows transcription
    • Methylation → protects inactive regions and prevents accidental transcription
  • Nucleosome modification:
    • Can slide up and down the DNA, changing which DNA is accessible for transcription
    • Can be remodeled
    • Can be temporarily removed

References

  1. Cooper GM (Ed.). (2000). The cell: a molecular approach. In Regulation of Transcription in Eukaryotes, 2nd ed. https://www.ncbi.nlm.nih.gov/books/NBK9904/
  2. Lodish H, Berk A, Zipursky SL, et al. (Ed.) (2000). Molecular cell biology. In The Three Roles of RNA in Protein Synthesis, 4th ed. https://www.ncbi.nlm.nih.gov/books/NBK21603/
  3. Christensen K, Hulick PJ. (2020). Basic genetics concepts: DNA regulation and gene expression. UpToDate. Retrieved April 15, 2021, from https://www.uptodate.com/contents/basic-genetics-concepts-dna-regulation-and-gene-expression 

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