constitutive expression vs. regulated expression
The control of gene expression * Cell type * Developmental stage * Environmental changes Genome transcriptome proteome Gene expression critical step
Gene regulation in prokaryotes (simple organism, common mechanism) Carbon source: glucose When glucose is low: lactose (disaccharide)
-galactosidase activity can be monitored by X-Gal Application: TA cloning
Growing in a new nutrient: enzyme level induction
Lactose is not an inducer
Two other proteins are also induced: Galactoside permease and thiogalactoside transacetylase (lactose transport) (detoxification) These proteins are associated with the same environmental change: Expression levels are regulated together! Such coordinated unit of gene expression: “Operon”
-galactosidase : z galactoside permease : y thiogalactoside transacetylase : a Mutant studies: z - y + a + no z; y and a are ok Mutant: z, y, a are expressed in the absence of lactose constitutive mutant
Mutant studies: Mutant: z, y, a are expressed in the absence of lactose constitutive mutant All three genes are regulated by a common element that is different from z, y, a themselves: “ i ” Wildtype inducible: i + z + y + a + Constitutive mutant: i - z + y + a + Is i activator or repressor?
Mutant studies: Episome: genetic material (plasmid) Diploid bacteria (by conjunction) Example: i + z - / (e)i - z + (chromosome/episome) Is this strain inducible or constitutive for -gal? Does i on the chromosome repress z on episome? How about i - z + / (e)i + z - ? A diffusible repressor is encoded by the i gene trans-acting factor
The operon model p: promoter sites o: operator site (regulatory DNA seq.) i: the repressor, binds to o z;y;a : polygenic or polycistronic RNA transcript
Lac repressor-DNA dimer/tetramer Monomer has helix- turn-helix: for major groove binding Consequence of lac repressor binding to operator?
Another example: pur repressor and pur operon pur repressor: * dimeric, 31% identical to lac repressor, similar in 3D structure * represses genes involved in purine biosynthesis * pur repressor binds DNA only when bound to a small molecule corepressor * Binds specific inverted-repeated sequence
pur operator: >20 19 operons, 25 genes
Dimer Inverted-repeated binding site Upregulate RNA pol. II 50x
Dimer of CAP bound to DNA
Sequence-specific DNA binding proteins and motif
Gene regulation depends on regulatory sites Symmetry in DNA sequence Protein symmetry
Many prokaryotic DNA-binding proteins have HTH motif Recognition helix: amino acids major groove bases Another helix: backbone interaction (dimeric)
Other proteins bind DNA through a pair of strands Methionine repressor
Eukaryotic DNA-binding motif: 1. homeodomain Similar to HTH Heterodimeric proteins asymmetric seq.