Two Levels of Enzyme Regulation
Genetic Level (Enzyme Induction)
Definition: Induction is stimulating the synthesis
of the enzyme’s messenger RNA.
Regulation is based on making
more enzyme protein
Substrate Level (covalent modification, allostery)
Substrate level regulates after the enzyme has been
synthesized. Regulation is based on activating
(or inhibiting) existing enzyme.
Covalent Modification:
Activate an inactive precursor by removing a
blocking peptide or adding or removing phosphate groups
Zymogen: An inactive enzyme precursor that
requires the removal of a blocking peptide to be active
Covalent Modification: Phosphorylation
Glycogen
phosphorylase b
(less active)
Glycogen
phosphorylase a
(more active)
The more active form is “a” and the less active
is “b”. Adding phosphate to the enzyme
stimulates enzyme activity.
Phosphorylation of Glycogen Enzymes
B
HO
OH
A
OPO3=
3PO
2ADP
(more active)
A
OH
(more active)
=O
2ATP
(less active)
HO
Glycogen
phosphorylase
Glycogen
synthase
2ATP
2ADP
B
=O
3PO
OPO3=
(less active)
The Hormone Connection
Rule: Enzymes that place phosphate groups on proteins
are called “protein kinases”.
Rule: Protein kinase activity is in part controlled by
the level of cyclic AMP within a cell.
Rule: Cyclic-AMP mediates the action of some hormones
ERGO
Rule: Phosphate group modifications explains how
hormones such as glucagon, insulin, and epinephrine
control enzyme activity in cells
Regulation of Human
Gene Expression
What do we know about the Human Genome?
Q: How many base pairs are in the human genome?
A: Approximately 3.2 billion, i.e., 3.2 x 109 or 3.2 gigabases
Q: Of this number how many are protein-encoding components?
A: Roughly 1.5 percent
Q: Have all of the genes been identified?
A: No, we are not even close
Q: What is left to do?
A: Gene products, i.e., functional mRNAs and proteins, need to be
identified, non-coding regulatory sequences need to be understood.
Q: What is are best estimate of the number of protein-coding genes?
A: Somewhere between 30,000 and 40,000
Q: What about the number of proteins in a human
A: Around 120,000
Q: How can there be more proteins than protein-coding genes?
A: Humans, more so than any other vertebrate species rely
on alternative splicing of a mRNA
Q: What to you mean by “alternative”
A: That means than one mRNA during processing can be
converted into more than one protein-coding mRNA
Q: And this is achieved by splicing?
A: It all depends on which exons are removed and which are
allowed to stay, or which sequences in introns make their way
into the final mRNA
Control of Eukaryotic Gene Expression
Rule: Regulation of Gene expression comes down to cellular
proteins interacting with DNA and RNA
Transcription level: enhancers, silencers
Post-Transcriptional level: spliceosomes, protein modification
What is Gene Silencing and why is this Important?
Rule: Opportune turning on or off of genes dictates the
spatial and chronological development of an organism
Cell, tissue and organ specificity
Cell development and differentiation
Programmed cell death (apoptosis)
Factoid: Practically every gene in eukaryotes is silent when
there is no activator to turn the gene on
Another Look at Mammalian Transcription
Rule: Transcription occurs on the surface of highly compact
DNA whose unwinding is essential for RNA to assemble
Heterochromatin (dense, inactive)
Euchromatin (open, active)
Barr bodies
Polytene
Chromosome
puffs
Histones and Nucleosome Structure
Acetylases and deacetylases
Left handed DNA in nucleosomes
RNA polymerase and + supercoiling
Nucleosomes
Polytene chromosome
(non-transcribed)
Drosophilia Chromosomes
RNA Synthesis occurs on chromatin,
not DNA
Polytene with
chromosome puffs
TBP TATA box binding Pro.
DNA binding domain
Enhancers
Act
Act
Activation domain (recruits other proteins)
Nucleosome
TAF
IIA TBP IIB
TATA
IIF
IIE
IIH
Pol II
mRNA
Turning a Gene on With a Hormone?
Indirectly
STATs: (Signal transducers and activators of transcription)
Cell signaling
Protein tyrosine kinases phosphorylate transcription
factors that penetrate the nucleus
Directly
STEROID HORMONES
Steriod hormone receptor proteins that penetrate the
nucleus as a protein-steroid complex and bind
directly to the promoter region on the DNA
Induce or repress expression of genes
Examples are the glucocorticoid receptor
Cell cycle is controlled by cyclins and
cyclin-dependent protein kinases (CDK’s)
How do genes control the cell cycle?
Key processes appear to be phosphorylations of nuclear proteins
by protein kinases
Cyclins and Cyclin kinases
Cdc2 kinase in yeast (first to be discovered)
34 kDa protein that phosphorylates threonine and serine residues
Activated by the cyclins at S and M phase of the cell cycle
Higher Eukaryotes
Cyclin-dependent kinases (CDKs)
Each transition of cell cycle is triggered by a unique cyclin/kinase
complex
e.g., CDK2 is required to enter S phase; cdc2, with cyclins A
and B regulate mitosis, cdc2 is phosphorylated to enter G2 and
dephosphorylated to continue mitosis
Cancer of the Retina
Promoter
Cyclin genes
Activates
E2F
P
Inactive
Rb
ADP
Cyclins
Cell proliferation
Stimulates
CDK
Cancer of the retina
CDK
ATP
E2F = a transcription factor that stimulates transcription of cyclin genes
Rb = retinoblastoma protein (tumor suppressor)
P53 and Cell Death (mutated in 50% of human cancers)
Promoter
pic1 gene
Activates
pic1
CDK
P
Stops mitosis
at G1
Promoter
p53
ADP
pic1-CDK
Apoptosis genes
CDK
ATP
Apoptosis proteins (caspases 3 and 9)
Destroys cells, prevents cancer cells from dividing
p53 = a transcription activator-tumor suppressor. It accumulates
when DNA is damaged. When phosphorylated, p53is a potent
activator of genes that stop the cell cycle or kill the cell. If
mutated (inactive) p53 allows damaged cells to replicate.
Final Exam for BICH 410 and 601
Monday, December 15, 2003
Room 108 Bio/Bio
10:30- 12:30
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