Regulation of Prokaryotic and Eukaryotic Gene Expression

This metabolic control occurs on two levels: A bacterium can tune its metabolism to the changing environment and food sources This metabolic control occurs on two levels: Adjusting activity of metabolic enzymes Regulating genes that encode metabolic enzymes

LE 18-20 Regulation of enzyme activity Regulation of enzyme production Precursor Feedback inhibition Enzyme 1 Gene 1 Enzyme 2 Gene 2 Regulation of gene expression Enzyme 3 Gene 3 Enzyme 4 Gene 4 Enzyme 5 Gene 5 Tryptophan

Operons: The Basic Concept In bacteria, genes are often clustered into operons, composed of An operator, an “on-off” switch A promoter Genes for metabolic enzymes

Polypeptides that make up enzymes for tryptophan synthesis LE 18-21a trp operon Promoter Promoter Genes of operon DNA trpR trpE trpD trpC trpB trpA Operator Regulatory gene RNA polymerase Start codon Stop codon 3¢ mRNA 5¢ mRNA 5¢ E D C B A Protein Inactive repressor Polypeptides that make up enzymes for tryptophan synthesis Tryptophan absent, repressor inactive, operon on

LE 18-21b_1 DNA mRNA Protein Active repressor Tryptophan (corepressor) Tryptophan present, repressor active, operon off

LE 18-21b_2 DNA No RNA made mRNA Protein Active repressor Tryptophan (corepressor) Tryptophan present, repressor active, operon off

Two Types of Negative Gene Regulation A repressible operon Is usually on binding of a repressor to the operator shuts off transcription The trp operon is a repressible operon An inducible operon Is one that is usually off a molecule called an inducer inactivates the repressor and turns on transcription the lac operon is an inducible operon, which contains genes coding for enzymes in hydrolysis and metabolism of lactose

LE 18-22a Regulatory gene Promoter Operator DNA lacl lacZ No RNA made 3¢ mRNA RNA polymerase 5¢ Active repressor Protein Lactose absent, repressor active, operon off

LE 18-22b lac operon DNA lacl lacZ lacY lacA RNA polymerase 3¢ mRNA 5¢ Permease Transacetylase Protein -Galactosidase Inactive repressor Allolactose (inducer) Lactose present, repressor inactive, operon on

Inducible enzymes usually function in catabolic (“breakdown”) pathways Explain to a neighbor why this makes sense. Repressible enzymes usually function in anabolic (“synthesis”) pathways Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor

Positive Gene Regulation Some operons are also subject to positive control through a stimulatory activator protein, such as catabolite activator protein (CAP) When glucose (a preferred food source of E. coli ) is scarce, the lac operon is activated by the binding of CAP When glucose levels increase, CAP detaches from the lac operon, turning it off

Lactose present, glucose scarce (cAMP level high): abundant lac LE 18-23a Promoter DNA lacl lacZ RNA polymerase can bind and transcribe CAP-binding site Operator Active CAP cAMP Inactive lac repressor Inactive CAP Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized

Lactose present, glucose present (cAMP level low): little lac LE 18-23b Promoter DNA lacl lacZ CAP-binding site Operator RNA polymerase can’t bind Inactive CAP Inactive lac repressor Lactose present, glucose present (cAMP level low): little lac mRNA synthesized

Summarize… Explain to a neighbor why bacteria regulate gene expression Give an example of how bacteria regulate gene expression

Eukaryotic Gene Regulation

How to prevent expression? Every cell in a multi-cellular eukaryote does not express all its genes, all the time (usually only 3-5%) Long-term control of gene expression in tissue = differentiation How to prevent expression? Regulation at transcription Regulation after transcription

Chromatin Regulation Chromatin remodeling allows transcription Chromatin = DNA + proteins Chromatin coiled around histones = nucleosomes Allows DNA to be packed into nucleus, but also physically regulates expression by making regions ‘available’ or not

Chromatin regulation can be small-scale (gene) or large scale (chromosome) Non-expressed = heterochromatin (condensed) Expressed = euchromatin (relaxed)

Changes to Chromatin (DNA) Methylation Methylating (adding methyl groups) to DNA bases, keeping them “tight” and “closed” – inaccessible to transcription. Histone Acetylation “Acetylating” histones (adding acetyl groups) promotes loose chromatin and permits transcription

Transcription Regulation What we know from prokaryotes: Several related genes can be transcribed together (ie. lac operon) Need RNA Polymerase to recognize a promoter region Why eukaryotes are different: Genes are nearly always transcribed individually 3 RNA Polymerases occur, requiring multiple proteins to initiate transcription

Transcription Regulation Con’t Typical prokaryotic promoter: recognition sequence + TATA box -> RNA Polymerase -> transcription Typical eukaryotic promoter: recognition sequence + TATA box + transcription factors -> RNA Polymerase -> transcription

RNA polymerase interacts w/promoter, regulator sequences, & enhancer sequences to begin transcription Regulator proteins bind to regulator sequences to activate transcription Found prior to promoter Enhancer sequences bind activator proteins Typically far from the gene Silencer sequences stop transcription if they bind with repressor proteins

Now, Can You: Explain why gene expression control is necessary in a eukaryotic cell? Describe how expression is regulated in before & during transcription? Tell me what differentiation is? Euchromatin? A silencer sequence? Explain how gene expression regulation is different in eukaryotes/prokaryotes?

Post-Transcription Regulation Have mRNA variation Alternative splicing: shuffling exons Allows various proteins to be produced in different tissues from the same gene Change the lifespan of mRNA Produce micro RNA that will damage mRNA, preventing translation Edit RNA & change the polypeptide produced Insert or alter the genetic code

Translation Regulation mRNA present in cytosol does not necessarily get translated into proteins Control the rate of translation to regulate gene expression How? Modify the 5’ cap Feedback regulation (build up of products = less translation)

Translation Regulation Con’t Modify the lifespan of proteins: Attach ubiquitin = target for breakdown via proteasome (woodchipper)

So… What are the ways that a cell can regulate gene expression AFTER transcription? How can the process of RNA splicing allow one pre-mRNA to produce 5 different proteins in 5 different tissues? And…

Can you accurately fill in this table?