Gene regulation V Biochemistry 302. March 6, 2006

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1 Gene regulation V Biochemistry 302 March 6, 2006

2 Common structural motifs associated with transcriptional regulatory proteins Helix-turn-helix Prokaryotic repressors and activators Eukaryotic homeodomain proteins Zinc-finger proteins C 2 H 2 class bind as monomers (TFIIIA, Zif268) C 4 (steroid receptors) and C 6 (Gal4) classes bind as dimers Winged-helix (forkhead) Bind as monomers HNF3 and TFs involved in early development Basic zipper (bzip) Bind as dimers GCN4, Fos, Jun, and other related members Helix-loop-helix Bind as dimers (basic N- term α helix, loop region, C-term amphipathic α helix c-myc, Max, Mad, and others Other motifs β-sheet motifs NFκB and MetR

3 Consequences of forming heterodimers (e.g. C 4 zinc-finger, bzip, HLH proteins) Expansion of number of potential DNA targets that a class of factors can bind. Different combinations of activation domains are brought together. Greater potential for regulation by inhibitory factors. Combinatorial complexity regulatory diversity Lodish et al. Molecular Cell Biology 3 rd edition

4 Structural motifs involved in DNA recognition and/or dimer formation Zinc finger Based on X- ray crystal structures Leucine (GCN4) bzip H-T-H Fig

5 Homeodomain, Engrailed C 2 H 2 Zn finger, Zif268 C 4 Zn finger, glucocorticoid receptor (monomer only) C 6 Zn finger, yeast Gal4 C 6 Zn finger, yeast Gal4 Winged helix or forkhead, HNF3 Coiled coil motif Lodish et al. Molecular Cell Biology 3 rd edition with citations therein Leucine bzip, yeast Gcn4 Leucine bzip, yeast Gcn4 H-L-H + Leu zipper, MAX

6 Other mechanisms of gene regulation (its not all about transcription initiation) Expression level Protein degradation (ubiquitination) Feedback regulation of transcription (gene deactivation) mrna degradation 3 UTR signals that protect against RNase degradation RNA interference by sirnas/mirnas Efficiency of mrna translation Stem loop signals in 5 -UTRs of some mrnas (e.g. IRE/IRP interaction in genes involved in iron metabolism) eif factor kinases (cell type and/or growth signal-dependent) Anti-sense mirnas Functional activity mrna processing: alternative poly(a) site selection, alternative splicing, RNA editing mrna localization (3 UTR zipcodes in actin mrnas) Post-translational modification (e.g. glycosylation, phosphorylation, sulfation, palmitoylation, farnesylation, acetylation, methylation.)

7 Feedback regulation of transcription 1: TFIIIA, a classic zinc finger protein, binds to internal control region (promoter). 2: Bound TFIIIA recruits TFIIIC. TFIIIA/C complex is stabilized by protein-protein and protein- DNA interactions. 3,4: PIC assembly completed by ATP-dependent recruitment of TFIIIB which allows binding of Pol III. (TFIIIB contains TBP but its exact role in 5S rrna transcription is unknown.) 5: PIC assembly is subject to negative feedback regulation by 5S rrna-tfiiia complex. +1 Specific for 5S gene, not needed for trnas Regulatory sequences lie within transcribed region Recycling of Pol III generates multiple transcripts Fig

8 Gene silencing by RNA interference (real biology or just a useful epi-genetic tool) Discovered in early 1990s as a defense mechanism against molecular parasites in plants RNA viruses Transposons Tool for epi-genetic studies Fire et al. (1998) Potent and specific genetic interference by double stranded RNA in C. elegans. Nature 391: Paddison et al. (2002) Short hairpin RNAs (shrnas) induce sequencespecific silencing in mammalian cells. Genes & Development 16: Two functional classes sirnas (artificial) strna/mirna (~250 mirna genes) Encoded as pre-mirna precursor, undergo elaborate processing Naturally-occurring mrna targets? (mirna lin-4 targets the TF lin-14 and controls lifespan in C. elegans) anti-sense sirna mirna mirna (imperfect complement) strnas are mirnas that appear transiently during development. ~22 nt

9 Important enzymes involved in sirnamediated mrna destruction Dicer: Double-stranded RNA dependent ribonuclease ~22 nt + cofactor protein: R2D2 RISC: RNA-induced silencing complex sirna strand w/ the most loosely base-paired 5 end ends up in the RISC complex Figure adapted from:

10 Hypothetical pathways of sirna and mirna-mediated gene silencing Guide complex? Rene F. Ketting and Ronald H.A. Plasterk EMBO reports 5: (2004) Two major regulatory sirna and mirna pathways: mrna destruction and repression of mrna translational

11 How Giardia intestinalis Dicer does it (member of RNase III family) Catalytic sites with two bound lanthanide metal (erbium, Er 3+ ) inhibitory cations (trivalent) Binds helical end of dsrna 3 overhang Asymmetric because Dicer cuts dsrna at staggered sites on each strand I. J. MacRae et al. Science 311: (2006)

12 Model of dsrna processing by Giardia Dicer based on crystal structure 65Å/2.6Å/bp = 25 bp Red and blue show acidic and basic protein surface. Front view Side view I. J. MacRae et al. Science 311: (2006)

13 Translational repression by mirna: importance of eif4e/cap & poly(a) tail Assay system: Translation of reporter mrna (Renilla-luciferase with 4 mirna target sites) transfected into HeLa cells (B) ± mirna (C,D). Note how substitution of 5 cap 7-methylguanosine with adenosine (A-cap) reduces repression efficiency of mirna. D. T. Humphreys et al. PNAS 102: (2005)

14 Conservation & tissue expression of mir-1 genes (mir-1-1 & mir-1-2) mir-1-1 promoter-lacz reporter mir-1-2 promoter:lacz reporter Y. Zhao et al. Nature 436: (2005) mir-1 gene promoters regulated by myogenic TFs: SRF, MEF2, and MyoD.

15 Model for regulation of cardiomyocyte growth and differentiation by mir-1 Zhao and coworkers developed a screening algorithm to identify putative targets of mir-1 predicted Hand2 gene. B. G. Bruneau Nature 436: (2005)

16 Repertoire of 5 - or 3 -UTR general and mrna-specific regulatory elements G. S. Wilkie et al. Trends in Biochem. Sci. 28: (2003)