Week 8 gene regulation in eukaryotes

8TH WEEK, BIO-1053 GENE REGULATION IN EUKARYOTES 8th week General Genetics-BIO1053 Chapter outline Overview of Eukaryotic Gene Regulation Control of Transcription Initiation Chromatin Structure and Epigenetic Effects Regulation After Transcription 8th week General Genetics-BIO1053 Overview of eukaryotic gene regulation Eukaryotes use complex sets of interactions • Regulated interactions of large networks of genes • Each gene has multiple points of regulation Themes of gene regulation in eukaryotes: • Environmental adaptation in unicellular eukaryotes • Maintenance of homeostasis in multicellular eukaryotes • Genes are turned on or off in the right place and time • Differentiation and precise positioning of tissues and organs during embryonic development 8th week General Genetics-BIO1053 Control of transcription initiation Three types of RNA polymerases in eukaryotes • RNA pol I – transcribes rRNA genes • RNA pol II – transcribes all protein-coding genes (mRNAs) and micro-RNAs • RNA pol III – transcribes tRNA genes and some small regulatory RNAs 8th week General Genetics-BIO1053 The three polymerases in eukaryotes These enzymes are eluted at different salt concentration during ion exchange chromatography Difference in their sensitivity to α-amanitin Pol I is very insensitive, Pol II is very sensitive, Pol III is intermediate sensitivity 8th week General Genetics-BIO1053 Comparison of three-dimensional structures of bacterial and eukaryotic RNA polymerases cis-acting elements: promoters and enhancers Promoters – usually directly adjacent to the gene • Include transcription initiation site • Often have TATA box: TATAA(T)AA(T) • Allow basal level of transcription Enhancers – can be far away from gene • Augment or repress the basal level of transcription trans-acting factors interact with cis-acting elements to control transcription initiation Direct effects of transcription factors: binding to DNA Indirect effect of transcription factors: protein-protein interactions Use of reporter genes to identify promoters and enhancers in eukaryotes RNA splicing helps regulate gene expression Sex lethal (Sxl) gene encodes a protein required for female-specific development In early embryos, Sxl is transcribed only in females RNA splicing helps regulate gene expression Later in development, Sxl gene is transcribed in both sexes Sxl protein regulates alternative splicing of its own mRNA RNA editing alters the sequences of pre-mRNAs The sequence of a pre-mRNA can be changed by a process, called RNA editing RNA editing occurs in the mitochondrial of protozoans and plants, also in chloroplasts In higher eukaryotes, RNA editing is rarer RNA editing of apo-B pre-mRNA apoB gene encodes two alternative forms of the serum protein apolipoprotein B: apoB-100 (≈ 5000 kDa) expressed in hepatocytes and apoB-48 (≈ 240 kDa) expressed in intestinal epithelial cells N-terminal domain (green): associates with lipids, C-terminal domain (orange) binds to LDL receptors on cell membranes Control of cytoplasmic polyadenylation and translation initiation Immature oocytes Mature oocytes CPE: cytoplasmic polyadenylation element CPEB: CPE-binding protein PABPI: poly (A)-binding protein CPSF: cleavage and polyadenylation specific factor PAP: poly (A) polymerase Pathways for degradation of eukaryotic mRNAs An ion-sensitive RNAbinding protein regulates mRNA translation and degradation IRE: ion response element IRE-BP: ion response element-binding-protein TfrR: transferrin receptor Some small RNAs are responsible for RNA interference (RNAi) Specialized RNAs that prevent expression of specific genes through complementary base pairing • Small (21 – 30 nt) RNAs • Micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) • First miRNAs (lin-4 and let-7) identified in C elegans • Nobel prize to A Fire and C Mello in 2006 Posttranscriptional mechanisms for gene regulation • mRNA stability and translation • May also affect chromatin structure Primary transcripts containing miRNA Most miRNAs are transcribed by RNA polymerase II from noncoding DNA regions that generate short dsRNA hairpins miRNA processing Drosha excises stem-loop from primary miRNA (pri-miRNA) to generate pre-miRNA of ~ 70 nt Dicer processes pre-miRNA to a mature duplex miRNA One strand is incorporated into miRNA-induced silencing complex (RISC) Two ways that miRNAs can down-regulate expression of target genes When complementarity is perfect: • Target mRNA is degraded When complementarity is imperfect: •Translation of mRNA target is repressed RNAi provides potential treatment for previously incurable diseases Use of RNAi in anti-cancer gene therapy miR-26a Triggers Cell-Cycle Arrest Adenoassociated viruses (AAV) exist as episomal DNA in the nucleus of cells A primary miR-26a transcript expressed by the viral vector is processed by Drosha/DGCR8 and is exported to cytoplasm where the pre-miRNA is further processed by Dicer/TRBP The guide strand is selected for entry into the RNA-induced silencing complex (RISC) The miR-26a guide strands pairs with sequences in the 3′ untranslated regions (UTRs) of target transcripts encoding the cyclins D2 and E2, thereby reducing their expression This results in the arrest of cells in G1 and inhibits proliferation of hepatocarcinoma cells 60 Posttranslational modifications of proteins Ubiquitination – covalent attachment of ubiquitin to other proteins targets those proteins for degradation by the proteosome Cascades of phosphorylation and dephosphorylation • Transmission of signals across the cell membrane to the nucleus • Sensitization – tissues exposed to hormones for long periods of time lose ability to respond to the hormone • Example: binding of epinephrine to β-adrenergic receptors on surface of heart muscle cells Phosphorylation and desensitization of β-adrenergic receptor Phosphorylation of receptor has no effect on its binding to epinephrine, but blocks its downstream functions ... eukaryotic gene regulation Eukaryotes use complex sets of interactions • Regulated interactions of large networks of genes • Each gene has multiple points of regulation Themes of gene regulation in eukaryotes: ...Chapter outline Overview of Eukaryotic Gene Regulation Control of Transcription Initiation Chromatin Structure and Epigenetic Effects Regulation After Transcription 8th week General Genetics-BIO1053... Chromatin remodeling and hypercondensation Epigenetic changes – changes in chromatin structure that are inherited from one generation to the next • DNA sequence is not altered 8th week General Genetics-BIO1053