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)