What makes cells different from each other? How do cells respond to information from environment? Regulation of: - Transcription - prokaryotes - eukaryotes - mrna splicing - mrna localisation and translation
Diversity of cell types and functions Red blood cells Hair cells in cochlea Cardiac muscle cells Skin cells Nerve cells What makes cells different is they make different proteins Some proteins made only in specific cell types: e.g., hemoglobin, insulin
Why do different cells make different proteins? They all have the same genes or do they? How do we know that s the case? Can clone a new animal from many different types of cells: Each cell has all genes necessary to make whole animal (and all cell types within it) Cloning => all cells have same genes All cells have same genes but not all genes expressed in each cell Housekeeping genes: - expressed in all cells (e.g., enzymes for cellular metabolism, structural proteins) Tissue-specific genes: - expressed in just some cell types (e.g., haemoglobin, insulin, liver enzymes, neurotransmitter receptors, etc.) Each cell type has a characteristic profile of expression of different subsets of the 30,000 genes in the genome
How is production of proteins regulated? How is each protein regulated? How is the regulation of all proteins coordinated? (differentiation) How do cells know which proteins to make? (development) How is protein expression modulated? (cellular metabolism / response to environment) Various steps in protein expression can all be regulated: - Transcription (in which cells or under what conditions does mrna get made for gene X?) - mrna splicing (many mrnas must be spliced together from separate exons to make mature message). - mrna localisation and translation (where and when do you make protein X from the mrna?) Core principles: Regulatory proteins bind control elements in DNA or RNA Control elements defined by specific sequences Both positive and negative regulation Mutations in either regulatory proteins or control elements can cause defects in regulation
Fundamental principles of differential gene expression first worked out in bacteria in the study of the response to nutrient availability Basic principles apply to all organisms and to different mechanisms of regulation Differential gene regulation: the lac operon Escherichia coli: preferred food source is glucose Will grow on other sugars like lactose Break it down to glucose and galactose Induction of β-galactosidase β-galactosidase protein normally hardly made at all (in presence of glucose and absence of lactose) But, in absence of glucose and presence of lactose: 10,000-fold induction in levels of β-galactosidase:
Growth on lactose depends on three enzymes: β-galactosidase (lacz) Permease (laca) Thiogalactoside transacetylase (lacy) Each encoded by a different gene, but: (i) Genes are tightly linked on chromosome (ii) All are induced coordinately (iii) Ratio of Z:A:Y proteins remains constant Conclude: under a common control system (an operon ) Lac genes are co-transcribed in a single mrna promoter Three proteins are translated separately How does the induction occur? How is the expression of these genes regulated in response to environmental conditions?
How was this discovered? Jacob and Monod (1961) isolated mutant strains of E. coli with altered growth on lactose or altered induction of lac genes: Wild-type Growth on Biochemical gene Mutant lactose phenotype z + z - - no galactosidase y + y - - no permease a + a - - no transacetylase o + o c + constitutive - all enzymes always present i + i - + constitutive i s - repressed (non-inducible) Dominance relationships and complementation Used an episome F-lac to make a partial diploid (a heterogenote) Test whether mutation only has effect on genes linked to it on the DNA: - A mutation in a regulatory element should only affect expression of genes right next to it ( in cis ) - A mutation in a gene encoding a regulatory protein should affect expression of genes on another chromosome ( in trans )
F episome complementation I makes some kind of diffusible substance (LacI repressor protein) The operator mutations only affect the expression of genes with which they are linked i.e., they only work in cis O does NOT make a diffusible substance
Explanation of mutants Mutant Lactose Explanation z - - no galactosidase y - - no permease a - - no transacetylase o c constitutive Mutation in operator sequence - repressor can not bind i - constitutive Mutation in repressor - it cannot bind operator i s - Mutation in repressor - it cannot bind lactose (always binds operator) I s mutations block binding of lactose to repressor (always off)
Lac operon also positively regulated Efficient binding of RNA polymerase to promoter requires another protein: - CAP (catabolite-activating protein) Binds to a specific sequence upstream of promoter Catabolite repression (inhibiting the activator) Induction of lac operon allows cell to make glucose from lactose But don t need to do that when have glucose around as well (don t need to make more glucose) Activator shut off when glucose high CAP must be bound to camp to be active Glucose inhibits production of camp by adenylyl cyclase Known as catabolite repression because high levels of catablolite (glucose) represses transcription Lac operon also positively regulated CAP
O 3 O 2 O 1 General principles from study of lac operon: Gene expression controlled by binding of proteins to upstream regulatory elements (operators/enhancers) Each gene controlled by both activators and repressors Separate response elements for different proteins Mutations in regulatory proteins or in the DNA elements can alter expression Expression regulated in response to external signals (from environment or from other cells in embryo) Lac operon is example of regulation of three linked genes How do you coordinate regulation of larger sets of (unlinked) genes? Will examine this in fruitfly Drosophila melanogaster