The BIG Questions…
•	How are genes turned on & off 
in eukaryotes?
•	How do cells with the same genes differentiate to perform completely different, specialized functions?
Evolution of gene regulation
•	Prokaryotes
•	single-celled
•	evolved to grow & divide rapidly
•	must respond quickly to changes in external environment
•	exploit transient resources
•	Gene regulation
•	turn genes on & off rapidly
•	flexibility & reversibility
•	adjust levels of enzymes 
for synthesis & digestion 
Evolution of gene regulation
•	Eukaryotes
•	multicellular
•	evolved to maintain constant internal conditions while facing changing external conditions
•	homeostasis
•	regulate body as a whole
•	growth & development
•	long term processes
•	specialization
•	turn on & off large number of genes
•	serve the needs of the body as a whole rather than the needs of individual cells
Points of control
•	The control of gene expression can occur at any step in the pathway from gene to functional protein
1.	unpacking DNA
2.	transcription
3.	mRNA processing
4.	mRNA transport
•	out of nucleus
•	through cytoplasm
•	protection from degradation
5.	translation
6.	protein processing
7.	protein degradation 
1. DNA packing
How do you fit all that DNA into nucleus?
•	DNA coiling & folding
•	double helix
•	nucleosomes
•	chromatin fiber
•	looped domains
•	chromosome
Nucleosomes 
•	“Beads on a string”
•	1st level of DNA packing
•	histone proteins 
•	8 protein molecules
•	many positively charged amino acids 
•	arginine & lysine
•	bind tightly to negatively charged DNA
DNA packing as gene control
•	Degree of packing of DNA regulates transcription
•	tightly wrapped around histones 
•	no transcription
•	genes turned off
DNA methylation
•	Methylation of DNA blocks transcription factors 
•	no transcription
	       genes turned off
•	attachment of methyl groups (–CH3) to cytosine 
•	C = cytosine
•	nearly permanent inactivation of genes
•	ex. inactivated mammalian X chromosome = Barr body
Histone acetylation
2. Transcription initiation
•	Control regions on DNA
•	promoter
•	nearby control sequence on DNA
•	binding of RNA polymerase & transcription factors
•	“base” rate of transcription
•	enhancers
•	distant control 
sequences on DNA
•	binding of activator 
proteins
•	“enhanced” rate (high level) 
of transcription
Model for Enhancer action
•	Enhancer DNA sequences 
•	distant control sequences
•	Activator proteins 
•	bind to enhancer sequence & stimulates transcription
•	Silencer proteins 
•	bind to enhancer sequence & block gene transcription
Transcription complex
3. Post-transcriptional control
•	Alternative RNA splicing
•	variable processing of exons creates a family of proteins
4. Regulation of mRNA degradation
•	Life span of mRNA determines amount of protein synthesis
•	mRNA can last from hours to weeks
RNA interference
•	microRNAs (miRNA)
•	short segments of RNA (21-28 bases)
•	bind to mRNA
•	create sections of double-stranded mRNA
•	“death” tag for mRNA
•	triggers degradation of mRNA
•	cause gene “silencing”
•	post-transcriptional control, but still effectively turns off the gene = no protein produced
Action of miRNA
5. Control of translation
•	Block initiation of translation stage 
•	regulatory proteins attach to 5’ end of mRNA 
•	prevent attachment of ribosomal subunits & initiator tRNA
•	block translation of mRNA to protein
6-7. Protein processing & degradation
•	Protein processing
•	folding, cleaving, adding sugar groups, targeting for transport
•	Protein degradation
•	ubiquitin tagging
•	proteasome degradation
Ubiquitin
•	“Death tag”
•	mark unwanted proteins with a label 
•	76 amino acid polypeptide, ubiquitin
•	labeled proteins are broken down rapidly in "waste disposers"
•	proteasomes
Proteasome 
•	Protein-degrading “machine”
•	cell’s waste disposer
•	breaks down any proteins 
into 7-9 amino acid fragments
•	cellular recycling