Riboswitches and Gene Regulation in Development (Lect. 19)

Ras pathway

1. a growth factor (e.g., EGF) binds its receptor, bringing 2 chains together 2. chains phosphorylate each other 3. phosphorylation attracts Grb2, which binds SOS 4. SOS binds Ras (GTPase), triggering GDP to GTP change (activation of Ras) 5. Ras activates a kinase at top of MAPK kinase cascade 6. MAPK (final kinase in cascade) phosphorylates transcription activators 7. activators enter nucleus, bind DNA and activate transcription DRAW

three strategies for signal transduction in gene expression

1. a ligand binds a receptor, initiating a kinase cascade causes phosphorylation of DNA-binding protein in nucleus 2. after binding ligand, receptor releases a DNA-binding protein into nucleus 3. after binding ligand, receptor cleaves off a cytoplasmic domain, which goes to the nucleus and associates with DNA-binding protein

typical riboswitch configuration in bacteria

1. aptamer 2. expression platform in 5' UTR, upstream of ORF

JAK/STAT pathway

1. cytokine binds receptor 2. 2 chains come together and JAK kinases on each phosphorylate each other (JAK and receptor) on tyrosines 3. after 2 STATs dock onto specific phosphorylated tyrosines on receptor (SH2 domains recognize), JAKs phosphorylate STATs 4. STATs dissociate and dimerize 5. STATs translocate to nucleus, bind DNA and activate transcription

types of cargo-carrying motors in the cell

1. kinesins walk along microtubules 2. myosins walk along actin filaments 3. dyneins also walk along microtubules

classes of ligand recognition structures

1. multihelical junctions a. junction is positioned centrally and joins P1 with other stems b. inverse junction architecture - stem folds back 2. pseudoknot folds

cellular "highways" for transporting cargo

1. polymers made of actin (i.e., actin filaments) 2. polymers made of tubulin (i.e., microtubules) 3. intermediate filaments

three strategies for initiating differential gene activity during development

1. signaling through diffusion of a secreted molecule (i.e., morphogen) e.g., Shh in development of neural tube 2. mRNA localization 3. cell-to-cell contact e.g., Notch signaling in skin-nerve regulatory switch

two general mechanisms for signal transduction in gene expression

1. signals that move things in and out of the nucleus e.g., JAK/STAT and Ras pathways in mammalian cells 2. signals that directly interact with transcription factors e.g., galactose and Gal80

discovery of the riboswitch

2002 generally, proteins recognize small molecules and bind to DNA or RNA to control expression of relevant genes BUT, no protein targets discovered for inhibition of biosynthetic genes for B1, B2 and B12 by thiamine

Ras pathway and disease

Ras mutations very common in cancer (especially pancreatic)

SAM riboswitch

SAM = S-adenosylmethionine functions to regulate genes in methionine biosynthesis 1. transcriptional terminator 2. sequesters ribosome binding sequence (RBS)

cytokine

a small protein secreted by one cell that influences another cell

diversity of riboswitch ligands in bacteria

a. anions b. metals c. purines and derivatives d. cofactors and derivatives e. amino acids

riboswitch mechanisms of gene control in bacteria

a. transcription termination Rho-dependent or independent b. transcription anti-termination c. translation inhibition d. translation activation

class of riboswitch structure and ligand structure...

are NOT correlated

usually, the binding pockets of riboswitches have...

conserved nucleotides a. conserved H-bonds with unpaired nucleotides b. stacking interactions c. Mg2+ used to compensate for any negative charge of the ligand

neural tube development

diffusible morphogen Shh dictates neuron type (different concentrations) in neural tube of developing embryo

Notch/Delta signaling

example of cell-to-cell contact in regulating development 1. neural cells express Delta do NOT express neuronal repressor genes (Notch target genes; repressed by Su(H)) 2. Delta activates Notch on surrounding cells 3. Notch is cleaved 4. cleaved region released into nucleus, turns Su(H) into activator instead of repressor 5. neuronal repressor genes activated in epidermal cells

GAL1 gene regulation

galactose molecule binds Gal80, unmasking Gal4 transcriptional activator and promoting transcription of GAL1 GAL1 encodes enzyme that metabolizes galactose

Ash1 in development

in yeast, mRNA encoding Ash1 repressor is localized to bud to prevent a switch in mating type myosin-driven movement along actin filaments

mechanism of mRNA localization in development

mRNA moved around as cargo, carried by cytoskeletal motor proteins (which move other stuff as well)

riboswitch

part of an mRNA that binds a metabolite and regulates gene expression in cis

expression platform

part of riboswitch that changes its folding pattern upon metabolite binding and controls gene expression DRAW

aptamer

part of riboswitch that selectively binds metabolites

P1

regulatory helix of riboswitches

structural principles of ligand recognition by riboswitches

tight binding pockets in 3D space that are the perfect shape for their ligand NO uniform metabolite recognition feature common to ALL riboswitches

Hox proteins

transcription factors that control body patterning and gene expression as a function of location in the body e.g., Ubx represses wing genes where haltere develops

cobalamin riboswitch

translation initiation controlled by ligand binding (stabilizes kissing loop interaction)

to form complex spatial conformations, riboswitches...

use (1) multihelical junction and (2) pseudoknot fold motifs as building blocks

general themes for JAK/STAT and Ras pathways

when not active, many DNA-binding activators (and repressors) are held in the cytoplasm the signal (like a cytokine from outside the cell) causes the activator (or repressor) to move into the nucleus, where it acts as (or on) a transcription factor (usually on initiation)