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PERSISTENCE: Mechanisms underlying the “Central Dogma” Annoucements: • Lab write-up due Thurs 5/17; Midterm will available xxx • Please fill out house ombuds surveys DKN • Where are we in the course? • What is the central dogma and why does it matter? • What important departures are there from the central dogma? • How can we leverage this understanding to do something meaningful? MMN • …How does the Central Dogma function in the Eukarya? Setting the stage – a major challenge of the eukaryotic cell – The Central Dogma is carried out in: - the nucleus, where the DNA is housed –TRANSCRIPTION - the cytoplasm, where the ribosomes occur - TRANSLATION Consequence? In Bacteria and Archaea – translation can begin before transcription ends In Eukarya – for most circumstances, a physical impossibility Chromosome – microscopically-visible elements of the cell, contain the DNA 100,000 to 10,000,000,000 bp With some exceptions: in Bacteria and Archaea – one or two, circular in Eukarya – linear, often as diploid # of chromosomes varies greatly, no correlations with phylogeny: plants – 5 (some flowering plants) to 1200 (fern) animals – smallest – jack jumper ant-- 2 in females 1 in males largest – rat spp. 92 humans – 46 Chromatin – DNA and proteins that make up the chromosomes Video of epigenetic changes to chromatin – http://www.youtube.com/watch?v=Tj_6DcUTRnM&feature=related Levels of Organization of the Chromatin/ Chromatin Packing: DNA HELIX Genes under active transcription add histones Active chromosome Less active genes add histones Inactive during replication add scaffolding proteins add histones (+ charged) 146 bp wrapped dna/histone core\ 60 bp linkers (proteins rich in Arg/Lys thus + and bind to negatively charged phosphates of DNA) 6 nm 12 nm GENE STRUCTURE AND TRANSCRIPTION/TRANSLATION IN EUKARYA transcription factor (a must)/ polymerase binding DNA transcription start start codon (ATG) Promoter 5’ UTR INTRON Transcription stop stop codon (TGA) EXON EXON transcription 5’ cap PreRNA INTRON INTRON EXON 5’ UTR INTRON EXON INTRON INTRON EXON EXON 3’ UTR polyA tail 3’ UTR splicing (alternative) RNA 5’ UTR Open reading frame 3’ UTR translation protein Bacteria Archaea Eucarya Basic differences among domains (Bacteria/Archaea/Eukarya) in transcription, i.e., usually: Eukarya: - Multiple polymerases with dedicated functions - Extensive post-transcriptional modifications - More regulatory sequences Will mitochondria and plastids transcribe DNA like Bacteria or like Eukarya? Why? Bacteria Archaea Transcriptional differences among domains, i.e., usually: Eucarya Coordinately controlled genes are: Operons Control elements found Bacteria/Archaea linked + once/operon Eukarya unlinked (same control elements) once/gene Control usually negative (repressors needed to turn gene off) usually positive (activator needed to turn gene on) mRNA 1 mRNA/operon short-lived 1 mRNA/gene variable (short/long-lived) [SHORT SUMMARY] Transcription/Translation in Eukarya: Multiple steps: A. must de-condense (loosen) the chromatin (protein mediated) B. transcription factors must bind to DNA C. polymerase binds to transcription factor (not directly onto DNA) to get actual transcription (multiple controls here) D. transcript gets modified (spliced, 3’ polyadenylation, 5’ cap) E. mature mRNA travels out to the cytoplasm where it makes a single protein REGULATION The process is regulated at every level: A. Transcriptional – weak and strong polymerase binding; enhancers/repressors B. Post-transcriptional – splicing, alternative splicing, different polyadenylation sites C. Translational – initiation (+/- 5’ cap; activity of initiation factors); variable lifetime of the RNA D. Post-translational – maturation of protein, phosphorylation, glycosylation Take home messages: How does the Central Dogma function in the Eukarya? - The presence of a nucleus has profound effects on the processes of transcription and translation. - Eukarya have more layers of regulation on all facet so the Central Dogma