BIOL 3301: Ch 15 Gene Regulation in EUkaryotes I: General Features of Transcriptional Regulation

gene expression

• process by which the information within a gene is used to synthesize RNA and polypeptides, and to affect the properties of cells and the phenotype of multicellular organisms

gene regulation

• the level of gene expression can vary under different conditions

importance of gene regulation

• respond to environmental changes such as nutrient availability, stress, etc.

• produce different cell types in multicellular species

• facilitate changes during development

  • some genes are only expressed during embryonic stages, whereas others are only expressed in the adult

eukaryotic regulation of gene expression

• transcription

  • regulatory transcription factors- activate or inhibit transcription

    • activators or repressors

combinatorial control

• most eukaryotic genes are regulated by many factors

• common factors contributing to ? are

  • 1 or more activator proteins may stimulate transcription

  • 1 or more repressor proteins may inhibit transcription

  • activators and repressors may be modulated by binding of small effector molecules, protein-protein interactions, and covalent modifications

transcription factors

• proteins that influence the ability of RNA polymerase to transcribe a given gene

• 2 main types:

  • general transcription factors

    • required for RNA pol to bind to the core promoter and for its progression to the elongation stage

    • necessary for basal transcription < very low transcription rate, usually can’t even be detected

  • regulatory transcription factors

    • serve to regulate transcription rate of target genes

    • they influence the ability of RNA pol to begin the transcription of a particular gene

general transcription factors

• required for RNA pol to bind to the core promoter and for its progression to the elongation stage

• necessary for basal transcription < very low transcription rate, usually can’t even be detected

regulatory transcription factors

• serve to regulate transcription rate of target genes

• they influence the ability of RNA pol to begin the transcription of a particular gene

domains

• regions in transcription factor proteins that have specific functions

  • DNA binding

  • binding site for small effector molecules

  • dimerization

    • ex., in many proteins, two identical subunits can bind to each other to form a functional dimer.

motif

• a domain, or a portion of a domain that has a very similar structure in many different proteins

• a polypeptide sequence that binds DNA that has a particular fx (e.g., DNA binding ? to help the protein grab on to the DNA).

  • domain

    • regions in transcription factor proteins that have specific functions: DNA binding, binding site for small effector molecules, dimerization

helix turn helix motif

• The motifs are special shapes or patterns in the transcription factors that allow them to bind to the DNA

• It’s like two sticks (α-helices) connected by a short bend (turn). The sticks help the protein bind to DNA.

helix loop helix motif

• The motifs are special shapes or patterns in the transcription factors that allow them to attach to the DNA.

• This motif looks like a small loop connecting two sticks. Two of these motifs can pair up and help the protein grab onto DNA.

regulatory domain

• in transcription factors, the ? can bind to specific DNA sequences or to other proteins (co-factors) to either activate or repress the transcription of genes

regulatory elements, control elements, regulatory sequences

• DNA sequences within enhancers that control gene activity by binding to regulatory transcription factors (activators or repressors)

  • (activators/repressors) regulatory transcription factors binding to ? affects the (increase/decrease) transcription of an associated gene

• cis regulatory elements

enhancer

• a DNA region, usually 50-1000 bp in length, that contains one or more of the regulatory elements/control elements/regulatory sequences

activator

• regulatory protein (regulatory transcription factor) that increases the rate of transcription

  • binds to regulatory element in enhancer

repressor

• regulatory protein/regulatory transcription factor that decreases the rate of transcription

  • binds to regulatory element in enhancer

activators

• proteins (regulatory transcription factor) bind to regulatory elements (enhancers) far from the gene's core promoter.

• DNA loops bring the activator close to the core promoter and preinitiation complex.

• This interaction increases RNA transcription by helping the transcription machinery start.

repressors

• proteins (regulatory transcription factor) can bind to enhancers and block the preinitiation complex.

• This prevents the transcription machinery from starting or completing RNA transcription, thus inhibiting transcription.

up regulation

• can be 10 to 1000 fold

• the binding of an activator (regulatory transcription factor) to an enhancer increases the rate of transcription

down regulation

• binding of a repressor (regulatory transcription factor) that inhibits transcription

enhancers

• orientation independent/bidirection

  • can function in forward (5’→3’) or reverse (3’→5’) orientation

• location

  • some can be close to the promoter, just a few hundred nucleotides upstream

  • However, they are often further away from promoter, 1000s or even 100,000 nucleotides away from the gene they regulate.

  • Enhancers can be downstream from the promoter or even within introns of the gene.

ways to modulate fx of regulatory transcription factors

• binding a small effector molecule (e.g., hormone)

  • small molecule binds RTF, changing its ability to bind DNA

• protein-protein interaction

  • RTF can form interactions with each other (e.g., dimerization), influencing their fx and DNA binding

• covalent modifications (e.g., phosphorylation)

  • chemical changes like phosphorylation (adding phosphate groups) can alter the fx of transcription factors

  • e.g., When it is phosphorylated, it can bind to DNA and activate transcription

RTF binds small effector molecule

• binding a ? (e.g., hormone)

  • small molecule binds RTF, changing its ability to bind DNA

• way to modulate fx of regulatory transcription factors

protein-protein interaction

• RTF can form interactions with each other (e.g., dimerization), influencing their fx and DNA binding

• ways to modulate fx of regulatory transcription factors

covalent modification

• chemical changes like phosphorylation (adding phosphate groups) can alter the fx of transcription factors

• e.g., When it is phosphorylated, it can bind to DNA and activate transcription

• ways to modulate fx of regulatory transcription factors

steroid receptors

• regulatory transcription factors that respond to steroid hormones

  • steroid hormone binds the regulatory transcription factor

  • ultimate action of steroid hormone is to affect gene transcription

steroid hormones

• these hormones bind to the regulatory transcription factor

  • purpose is to affect gene transcription

• secreted by endocrine glands into the bloodstream

  • then taken up by cells that respond to the hormone

  • cells respond to ? hormones in different ways

    • glucocorticoids (? hormone)

      • influence nutrient metabolism in most cells

      • promote glucose utilization, fat mobilization, and protein breakdown

glucocorticoid response elements GRE

• DNA sequences within enhancers

  • located near dozens of different genes, allowing the hormone (glucocorticoid) to activate many genes

• consists of 2 sequences that are close together

  • 5’ AGRACA 3’

  • 3’ TCYTGT 5’

• glucocorticoid hormones enter cell and bind to the glucocorticoid receptor subunits that dimerize, enter the nucleus, bind to ? and activate gene transcription

glucocorticoids

• steroid hormones

• influence nutrient metabolism in most cells

• promote glucose utilization, fat mobilization, and protein breakdown

• enter cell and bind to the glucocorticoid receptor subunits that dimerize, enter the nucleus, bind to a Glucocorticoid Response Element (GRE) and activate gene transcription

glucocorticoid hormone action

• Glucocorticoid hormone enters the cell and binds to the glucocorticoid receptor.

• The binding of the hormone causes the release of HSP90 (a heat shock protein).

• This release exposes a nuclear localization signal (NLS) on the receptor.

• The two glucocorticoid receptors with the hormone dimerize and enter the nucleus.

• Inside the nucleus, the dimer binds to the Glucocorticoid Response Element (GRE) on the DNA.

• The binding of the receptors to the GRE activates transcription of a nearby gene.

• GRE can be located far away from the target gene, even within an enhancer.

CREB cAMP response element-binding protein

• a regulatory transcription factor

• gets activated when cAMP (cyclic adenosine monophosphate) levels increase inside the cell

  • cAMP is a common signaling molecule

  • extracellular signaling molecules cause an increase in cytoplasmic [cAMP]

• binds to DNA at two adjacent sites called CREs (cAMP Response elements)

  • CRE consensus sequence

    • 5’-TGACGTCA-3’

    • 3’-ACTGCAGT-5’

• binding to the CRE allows ? to help activate transcription of target genes

cAMP response element CRE

• consensus DNA sequence

  • 5’-TGACGTCA-3’

  • 3’-ACTGCAGT-5’

• binds CREB protein, a transcription factor

  • CREB activated when cAMP levels rise

    • activated CREB boosts gene transcription

CREB protein action

• extracellular signaling molecules (like epinephrine) trigger cAMP production inside the cell

• cAMP acts as a second messenger

  • First messenger = signal from outside the cell (like a hormone)

  • Second messenger = small molecule inside the cell (like cAMP) that carries the message and activates cellular responses.

• cAMP activates protein kinase A (PKA)

  • PKA phosphorylates CREB protein, activating it

  • phosphorylated CREB binds co-activator CREB-binding protein CBP→ greatly increase adjacent gene transcription

CREB protein pathway to transcriptional activation

• A signal molecule (like a hormone) binds to a membrane receptor, activating a G protein.

• The G protein activates adenylyl cyclase, which makes cAMP from ATP.

• cAMP activates protein kinase A (PKA).

• PKA enters the nucleus and phosphorylates the CREB protein.

• Phosphorylated CREB binds to CBP (a coactivator) and activates transcription of the target gene at the CRE site.