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 Glycogen phosphorylase b phosphorylase a (less active) (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 phosphorylase OH HO B OPO3= =O3PO A 2ATP 2ADP (less active) (more active) Glycogen synthase HO OH A OPO3= =O3PO B 2ATP 2ADP (more active) (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 Barr bodies Polytene Heterochromatin (dense, inactive) Euchromatin (open, active) Chromosome puffs Histones and Nucleosome Structure Nucleosomes Acetylases and deacetylases Left handed DNA in nucleosomes RNA polymerase and + supercoiling

RNA Synthesis occurs on chromatin, not DNA Polytene chromosome (non-transcribed) Polytene with chromosome puffs Drosophilia Chromosomes RNA Synthesis occurs on chromatin, not DNA TBP TATA box binding Pro. TATA Enhancers Nucleosome DNA binding domain Act Act Activation domain (recruits other proteins) TAF IIF IIE IIH TBP IIA IIB 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 E2F Cyclins Cell proliferation Inactive Rb Cyclin genes Promoter Activates E2F Cyclins Cell proliferation P Stimulates CDK Inactive Rb Cancer of the retina ADP ATP CDK 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) pic1 gene Promoter Activates pic1 pic1-CDK Stops mitosis at G1 CDK P Apoptosis genes Promoter p53 ADP ATP CDK 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 Bring Scantron Bring Calculator