Eukaryotic Gene Regulation

1. Eukaryotic Gene Regulation • Four levels of gene regulation in eukaryotes include: • Transcriptional • Posttranscriptional • Translational • Posttranslational

2. Transcriptional Control • Dependent upon organization of chromatin • euchromatin (genetically active) • heterochromatin (genetically inactive) • Euchromatin allows access to the DNA (so the genes can be expressed)

3. Transcriptional Control • Transcriptional control involves DNA-binding proteins called transcription factors. • These bind to the promoter then bind RNA polymerase. • Transcription factors are always present, but usually require activation.

4. Transcription Factors • Activation usually occurs through the addition or removal of phosphate group to the molecule • Can be phosphorylated by a kinase (adds a phosphate group to a molecule) • Can be dephosphorylated by phosphatase (removes a phosphate group) • Kinases and phosphatases are signaling proteins

5. Posttranscriptional Control • This begins once there is an mRNA transcript. • Involves differential mRNA processing (some exons may get spliced out before leaving the nucleus) • Also involves length of time that it takes for mRNA to travel to cytoplasm.

6. Translational Control • Begins when processed mRNA reaches ribosome in cytoplasm. • The longer an active mRNA molecule remains in cytoplasm, the more that the product is made. • Cap and poly-A tail may determine how long transcript remains active. • Hormones seem to cause stabilization of certain mRNA transcripts (ex. Prolactin helps stabilize milk production)

7. Posttranslational Control • Begins once a protein has been synthesized. • Feedback inhibition regulates metabolic pathways. • Some proteins are not active immediately after synthesis. Activation may require: • Folding (3D structure) occurs chemically and is aided by chaperones (proteins whose function is to assist other proteins in achieving proper folding) • Cleavage of portion • Addition of functional groups

8. =O CH3C- H H H-C- Functional Groups • Acetylation (addition of an acetyl group) • Glycosylation (addition of a glucose derivative) • Methylation (addition of a methyl group) • Phosphorylation (addition of a phosphate group) • Sulfation (addition of a sulfate group)

9. Gene Regulation Animation Control of gene expression in Eukaryotes

10. RNA interference (RNAi) • Previously known as “gene silencing” • Example of post-transcriptional gene regulation • First discovered in 1998 by Dr. Andrew Fire & Dr. Craig Mello using C. elegans (roundworm) • Step 1: Double-stranded RNA (dsRNA) introduced to the cell (usually via viral vector) - not too effective in mammals • Step 2: dsRNA gets processed into small interfering RNAs (siRNAs) by an enzyme called Dicer (ribonuclease - RNase)

11. RNA interference (RNAi) • Step 3: siRNAs assemble into complexes called RNA-induced silencing complexes (RISCs), unwinding (to become single stranded) in the process • RISC complexes include helicase & endonucleases • Step 4: siRNAs guide the RISCs to complementary RNA molecules where they cleave and destroy the target RNA, preventing translation • Used in biotechnology to lower natural levels of plant toxins

12. RNA interference (RNAi) RNAi animation

13. RNAi Problems • Delivery of RNAi drugs to the right targets • Avoiding the silencing “good” genes • Ensuring that the drugs stay active long enough to help the patient