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Transcript
___________________________________ D. Cell Specialization: Regulation of Transcription ___________________________________ ___________________________________ Cell specialization in multicellular organisms results from differential gene expression ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ D. CELL SPECIALIZATION: Regulation of Transcription 1. Chromosome, Gene and RNA Architecture 2. Cell-Specific Regulation of Chromosome Structure ___________________________________ ___________________________________ ___________________________________ 3. Cell-Specific Regulation of Transcription Activation ___________________________________ ___________________________________ ___________________________________ 1. Review of Chromosome, Gene and RNA Architecture a. Review of Chromatin Structure b. Chromosomal Gene Arrangement ___________________________________ ___________________________________ ___________________________________ c. Single Gene Components d. Nuclear RNA, mRNA and Protein e. Other RNA Molecules ___________________________________ ___________________________________ f. Fast review of Transcription ___________________________________ ___________________________________ ___________________________________ a. Review of Chromatin Structure • Chromatin is a complex of DNA and protein in the eukaryotic nucleus ___________________________________ • Loosely packed chromatin is called euchromatin ___________________________________ • Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions ___________________________________ • Histones are proteins that are responsible for the first level of DNA packing in chromatin ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Nucleosome (10 nm in diameter) DNA double helix (2 nm in diameter) Histones DNA, the double helix Histones ___________________________________ H1 ___________________________________ Nucleosomes, or “beads on a string” (10-nm fiber) ___________________________________ Histone tail ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Figure 4-65 Molecular Biology of the Cell (© Garland Science 2008) ___________________________________ b. Chromosomal Gene Arrangement ___________________________________ ___________________________________ Humans: 23 chromosome pairs ___________________________________ 3 billion bases ~24,000 genes ___________________________________ ___________________________________ ___________________________________ Figure 4-11 Molecular Biology of the Cell (© Garland Science 2008) ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Figure 4-15 Molecular Biology of the Cell (© Garland Science 2008) ___________________________________ ___________________________________ Genes can reside on either strand ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Figure 6-14 Molecular Biology of the Cell (© Garland Science 2008) ___________________________________ c. Single Gene Components Anatomy of a gene ___________________________________ ___________________________________ ___________________________________ ___________________________________ Exon means sequence that exits the nucleus ___________________________________ Intron means sequence that stays inside the nucleus ___________________________________ ___________________________________ d. Nuclear RNA, mRNA and Protein ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Figure 6-21 Molecular Biology of the Cell (© Garland Science 2008) ___________________________________ ___________________________________ ___________________________________ e. Other RNA Molecules ___________________________________ 1. The Translational Apparatus 2. Nuclear Effectors ___________________________________ 3. Cytosolic Effectors ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Table 6-1 Molecular Biology of the Cell (© Garland Science 2008) ___________________________________ ___________________________________ f. Fast review of transcription Templated Polymerization DNA molecule Gene 2 ___________________________________ Gene 1 Gene 3 Transcription: “To transcribe” to copy in the same language Translation: “To translate” to copy into a new language ___________________________________ DNA template strand TRANSCRIPTION ___________________________________ mRNA Codon TRANSLATION ___________________________________ Protein Amino acid ___________________________________ ___________________________________ ___________________________________ RNA Polymerase II Complex Does it All ___________________________________ ___________________________________ ___________________________________ ___________________________________ 12 Protein Subunits in Human ___________________________________ Figure 6-8a Molecular Biology of the Cell (© Garland Science 2008) ___________________________________ ___________________________________ • As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time • Transcription progresses at a rate of 40 nucleotides per second in eukaryotes ___________________________________ • The large subunit of RNA Pol II caps and polyadenylates the nascent nRNA ___________________________________ • The same large subunit of RNA also links to the splicosome to facilitate subsequent processing ___________________________________ ___________________________________ ___________________________________ Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings ___________________________________ ___________________________________ Multiple RNA Pol II molecules can read DNA simultaneously ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Figure 6-9 Molecular Biology of the Cell (© Garland Science 2008) ___________________________________ ___________________________________ • So, how do individual cells regulate which of the genes in their genome they will express? • Remember from Intro Bio that prokaryotes regulate expression through repressors/activators • Eukaryotes have more complex regulatory mechanisms ___________________________________ ___________________________________ • Histone modification regulates chromatin structure • DNA modification regulates promoter accessibility ___________________________________ • Epigenetic modification can be copied and inherited • Transcription factors regulate promoter activation ___________________________________ • Specialized transcriptional activities ___________________________________ ___________________________________ 2. Nucleosome and Histone Modification Regulates of Chromatin Structure • Chromatin is a complex of DNA and protein in the eukaryotic nucleus ___________________________________ ___________________________________ • Loosely packed chromatin is called euchromatin ___________________________________ • Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions ___________________________________ • Histones are proteins that are responsible for the first level of DNA packing in chromatin ___________________________________ ___________________________________ ___________________________________ Cell-specific control of chromosome structure Eukaryotic cells can systematically control which genes are available for expression. ___________________________________ ___________________________________ Our DNA is complexed 50:50 with proteins and is very highly regulated by enzymatic alterations of what is open and closed. ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Spontaneous nucleosome unwrapping ___________________________________ ___________________________________ Figure 4-28 Molecular Biology of the Cell (© Garland Science 2008) ___________________________________ ___________________________________ ATP-dependent nucleosome unwrapping ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Figure 4-29 Molecular Biology of the Cell (© Garland Science 2008) ___________________________________ Histones are covalently modified to control gene accessibility • The methylation and/or acetylation of either histones or the DNA itself determines what promoters are exposed. • Different cell types have different enzymes and, thus, different areas of protein and DNA are targeted for alteration ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Cell-specific control of chromosome structure 3 Stages of Transcription 1. Initiation 2. Elongation 3. Termination ___________________________________ ___________________________________ ___________________________________ (lysine amino acid residues) ___________________________________ ___________________________________ Acetylation promotes Initiation Methylation can go either way ___________________________________ ___________________________________ 3. The “Histone Code” Hypothesis ___________________________________ • Combinations of covalent modifications have specific information for the cell ___________________________________ – This DNA is newly replicated ___________________________________ – This DNA is damaged and needs repair – Express this DNA ___________________________________ – Put this DNA into heterochromatin storage ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Figure 4-43 Molecular Biology of the Cell (© Garland Science 2008) ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Figure 4-45 Molecular Biology of the Cell (© Garland Science 2008) ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Figure 4-46a Molecular Biology of the Cell (© Garland Science 2008) ___________________________________ ___________________________________ ___________________________________ b. Direct covalent modifications of DNA can also control expression from genes in the euchromatin ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Methylation of globin genes in human embryonic blood cells ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ c. Heritability: Epigenetic Memory OK. So a cell differentiates to become a blood vessel smooth muscle cell or fibroblast ...... ___________________________________ ___________________________________ ___________________________________ • How come all of its mitotic descendents don’t have to go through differentiation? – Trithorax proteins bind to open nucleosomes and keep them open. – Polycomb proteins methylate uneeded nucleosomes and then bind to them to keep them tight. – These effects can then be directly passed through mitotic cell division to the offspring. ___________________________________ ___________________________________ ___________________________________ ___________________________________ Two DNA methyltransferases are important in modifying DNA ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ d. Transcription factors regulate promoter activation – Core promoter made up of TATAbox and CpG islands • Site of RNA Pol II recruitment and activation ___________________________________ ___________________________________ • TF II family transcription factors bind RNA Pol II to core ___________________________________ – Tissue-specific TF are true transcriptional determinant for the cell type ___________________________________ • Bind to core promoter elements and distal enhancers • Create binding sites for TF II family TF and stabilize Transcription Initiation Complex ___________________________________ • TS-TF also recruit histone acetyltransferases to expose DNA ___________________________________ ___________________________________ ___________________________________ Tissue-Specific Transcription Factor Families ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Fig. 17-8 Promoter 5 3 TATA box Template 3 5 Start point Template DNA strand General Transcription factors 5 3 3 5 The Transcription Initiation Complex forms on every gene that gets expressed. Its presence there is really determined by the tissue specific transcription factors that bind to enhancer cis-elements. ___________________________________ ___________________________________ ___________________________________ RNA polymerase II Cell-Specific Transcription factors 5 3 3 5 5 RNA transcript Transcription initiation complex ___________________________________ ___________________________________ ___________________________________ RNA polymerase is stabilized on the promoter site of the DNA by transcription factors recruited by promoters and enhancers ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Fig. 17-7b Nontemplate strand of DNA Elongation RNA polymerase 3’ RNA nucleotides ___________________________________ ___________________________________ 3 end 5’ 5 Direction of transcription (“downstream”) Template strand of DNA Newly made RNA ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ The stability of the initiation complex determines how many transcripts ___________________________________ Figure 6-3 Molecular Biology of the Cell (© Garland Science 2008) ___________________________________ Tissue-specific transcription factors may bind different enhancers ___________________________________ ___________________________________ ___________________________________ ___________________________________ The pax-6 gene has four enhancers and is expressed exclusively in those four tissue types. ___________________________________ ___________________________________ ___________________________________ TS TFs can even control differentiation stability A really important idea in cell differentiation is that there must be a molecular mechanism that keeps a cell differentiated. ___________________________________ ___________________________________ ___________________________________ – The pax-6 gene has a Pax-6 site in its enhancer – When it is present the transcription rate is maximal – This mechanism is repeated in several cell types ___________________________________ ___________________________________ – A rare positive feedback loop ___________________________________ ___________________________________ e. Specialized transcriptional activities ___________________________________ • Only about 3-5% of RNA in a cell is mRNA ___________________________________ • Up to 80% of RNA is ribosomal RNA – As many as 10 million ribosomes per cell ___________________________________ – humans have 400 rRNA gene copies on 5 chromosome pairs (frogs have 1200) ___________________________________ – 4 eukaryotic subunits: 18S, 5.8S, 28S, 5S – First 3 from one gene with RNA Pol I – 5S is from a separate gene with RNA Pol III ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Figure 6-42 Molecular Biology of the Cell (© Garland Science 2008) ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Table 6-1 Molecular Biology of the Cell (© Garland Science 2008) ___________________________________ RNA Molecules and their RNA Polymerases ___________________________________ • Most snRNA and miRNA: Pol II ___________________________________ • tRNA, shRNA, snRNA 6, miRNA: Pol III • snoRNA often encoded in introns: Pol II ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________
