Eukaryotic Genomes: Organization, Regulation, and Evolution

1. Eukaryotic Genomes: Organization, Regulation, and Evolution Chapter 19

2. 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? 2

3. Organization

4. Prokaryote vs. eukaryote genome • Prokaryotes • small size of genome • circular molecule of naked DNA • DNA is readily available to RNA polymerase • control of transcription by regulatory proteins • operon system • most of DNA codes for protein or RNA • no introns, small amount of non-coding DNA • regulatory sequences: promoters, operators 4

5. Prokaryote vs. eukaryote genome • Eukaryotes • much greater size of genome • how does all that DNA fit into nucleus? • DNA packaged in chromatin fibers • regulates access to DNA by RNA polymerase • cell specialization • need to turn on & off large numbers of genes • most of DNA does not code for protein • 97% “junk DNA” in humans 5

6. Points of control • The control of gene expression can occur at any step in the pathway from gene to functional protein • unpacking DNA • transcription • mRNA processing • mRNA transport • out of nucleus • through cytoplasm • protection from degradation • translation • protein processing • protein degradation 6

7. Why turn genes on & off? • Specialization • each cell of a multicellular eukaryote expresses only a small fraction of its genes • Development • different genes needed at different points in life cycle of an organism • afterwards need to be turned off permanently • Responding to organism’s needs • homeostasis • cells of multicellular organisms must continually turn certain genes on & off in response to signals from their external & internal environment 7

8. DNA packing How do you fit all that DNA into nucleus? • DNA coiling & folding • double helix • nucleosomes • chromatin fiber • looped domains • chromosome from DNA double helix to condensed chromosome 8

9. Chromatin Structure • Based on successive levels of DNA packing • Eukaryotic DNA is precisely combined with a large amount of protein • Chromatin changes during the course of the cell cycle • Eukaryotic chromosomes contain an enormous amount of DNA relative to their condensed length • Helps to regulate gene expression, condense and release and form chromosomes

10. Nucleosomes • Proteins called histones • Are responsible for the first level of DNA packing in chromatin • Bind tightly to DNA • The association of DNA and histones • Seems to remain intact throughout the cell cycle • In electron micrographs • Unfolded chromatin has the appearance of beads on a string

11. 2 nm DNA double helix Histone tails His- tones 10 nm Histone H1 Linker DNA (“string”) Nucleosome (“bead”) Nucleosomes • Each “bead” is a nucleosome • The basic unit of DNA packing

12. 30 nm Nucleosome Higher Levels of DNA Packing • Interactions between the histone tails of the nucleosomes • Causes the nucleosomes to coil around each other • Degree of packing of DNA regulates transcription • tightly packed = no transcription • = genes turned off

13. Regulation

14. Gene Regulation • All organisms • Must regulate which genes are expressed at any given time • Each cell of a multi-cellular eukaryote • Expresses only a fraction of its genes • In each type of differentiated cell • A unique subset of genes is expressed • In interphase cells • Most chromatin is in the highly extended form called euchromatin • Genes within highly packed heterochromatin • Are usually not expressed

15. Signal NUCLEUS Chromatin Chromatin modification: DNA unpacking involving histone acetylation and DNA demethlation DNA Gene available for transcription Gene Transcription Exon RNA Primary transcript Intron RNA processing Tail mRNA in nucleus Cap Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Polypetide Cleavage Chemical modification Transport to cellular destination Active protein Degradation of protein Degraded protein Gene Regulation • Many key stages of gene expression • Can be regulated in eukaryotic cells • Each gene is regulated in its own particular way or ways

16. Regulation of Chromatin Structure • Histone Modifications- • Chemical modification of histone tails • Affect the configuration of chromatin and thus gene expression • Histone acetylation • Addition of acetyl group (-COCH3) Seems to loosen chromatin structure and thereby enhance transcription • DNA Methylation- addition of a methyl group (– CH3) reducing transcription in some species

17. 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

18. Post-Transcriptional Regulation

19. Poly-A signal sequence Termination region Proximal control elements Enhancer (distal control elements) Exon Intron Intron Exon Exon DNA Downstream Upstream Promoter Transcription Poly-A signal Exon Exon Intron Intron Exon Cleared 3 end of primary transport Primary RNA transcript (pre-mRNA) 5 Chromatin changes RNA processing: Cap and tail added; introns excised and exons spliced together Transcription Intron RNA RNA processing Coding segment mRNA degradation Translation mRNA P G Protein processing and degradation P P Start codon Poly-A tail Stop codon 3 UTR (untranslated region) 5 Cap 5 UTR (untranslated region) • Associated with most eukaryotic genes are multiple control elements • Segments of noncoding DNA that help regulate transcription by binding certain proteins

20. Transcription Factors • To initiate transcription • Eukaryotic RNA polymerase requires the assistance of proteins called transcription factors • Only when the complete initiation complex is assembled can the polymerase produce the complimentary strand • Some specific transcription factors function as repressors • To inhibit expression of a particular gene

21. Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Exons DNA Primary RNA transcript RNA splicing or mRNA Post- Transcriptional Regulation • RNA Processing- • In alternative RNA splicing different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns

22. Post-Transcriptional Regulation • mRNA Degradation • The life span of mRNA molecules in the cytoplasm • Is an important factor in determining the protein synthesis in a cell • Determines how long the mRNA will last in the cytoplasm and how many times the mRNA will be read • mRNA can last from hours to weeks

23. mRNA Degradation • microRNA (miRNA) • single stranded that binds to mRNA that fold back on themselves. • a dicer cuts the double stranded RNA into short fragments. One strand is degraded and the other either degrades or blocks translation • RNA interference (RNAi) • double stranded RNA injected into cell turns off a gene • Small interfering RNAs (siRNA) • bind to mRNA • create sections of double-stranded mRNA • “death” tag for mRNA • triggers degradation of mRNA • cause gene “silencing” • even though post-transcriptional control, still turns off a gene Small RNAs mRNA double-stranded RNA sRNA + mRNA mRNA degraded functionally turns gene off 23

24. Initiation of Translation • The initiation of translation of selected mRNAs • Can be blocked by regulatory proteins that bind to specific sequences or structures of the mRNA • Prevent ribosome from attaching to the mRNA • Translation of all the mRNAs in a cell may be regulated simultaneously • Plays a role in the translation of mRNA’s stored in egg cells

25. Post- Translational Regulation • After translation various types of protein processing, including cleavage and the addition of chemical groups, are subject to control • Proteasomes • Are giant protein complexes that bind protein molecules and degrade them (can breakdown all proteins into 7-9 amino acid fragments) • Mutations in proteasomes can lead to cancer

26. 1. transcription -DNA packing -transcription factors 2. mRNA processing -splicing 3.mRNA transport out of nucleus -breakdown by sRNA 4. mRNA transport in cytoplasm -protection by 5’ cap & poly-A tail 5. translation -factors which block start of translation 6. post-translation -protein processing -protein degradation -ubiquitin, proteasome 6 post-translation 4 5 translation mRNA transport in cytoplasm 1 transcription 3 mRNA transport out of nucleus 2 mRNA processing 26

27. Cancer

28. Cancer • Cancer results from genetic changes that affect cell cycle control • The gene regulation systems that go wrong during cancer play important roles in embryonic development • The genes that normally regulate cell growth and division during the cell cycle • Include genes for growth factors, their receptors, and the intracellular molecules of signaling pathways

29. Cancer • Oncogenes • Are cancer-causing genes • Proto-oncogenes • Are normal cellular genes that code for proteins that stimulate normal cell growth and division • An oncogene arises from a genetic change in a proto-oncogene that either increases the amount of protein produced or in the activity of the protein molecule

30. Proto-oncogene DNA Translocation or transposition: gene moved to new locus, under new controls Point mutation within a control element Point mutation within the gene Gene amplification: multiple copies of the gene Oncogene Oncogene New promoter Normal growth-stimulating protein in excess Hyperactive or degradation- resistant protein Normal growth-stimulating protein in excess Normal growth-stimulating protein in excess Mutations that change proto-oncogenes into oncogenes Cancer cells are often found to contain chromosomes that have been broken and rejoined incorrectly, translocating fragments from one chromosome to another. If the translocated proto-oncogene ends up near an active promotor, it may increase transcription, making it an oncogene.

31. Tumor Suppression • Tumor-suppressor genes (p53 is common) • Encode proteins that inhibit abnormal cell division • Mutation in these may contribute to the onset of cancer • Code for proteins that: • Repair damaged DNA • Control adhesion of cells to each other • Inhibit the cell cycle

32. Colon 1 Loss of tumor- suppressor gene APC (or other) 4 Loss of tumor-suppressor gene p53 2 Activation of ras oncogene Colon wall 3 Loss of tumor- suppressor gene DCC 5 Additional mutations Normal colon epithelial cells Larger benign growth (adenoma) Small benign growth (polyp) Malignant tumor (carcinoma) Cancer Development • Normal cells are converted to cancer cells • By the accumulation of multiple mutations affecting proto-oncogenes and tumor-suppressor genes • A multistep model for the development of colorectal cancer

33. Other Promoters of Cancer • Certain viruses • Promote cancer by integration of viral DNA into a cell’s genome • Individuals who inherit a mutant oncogene or tumor-suppressor allele • Have an increased risk of developing certain types of cancer