Chapter 18 Control of Gene Expression in Eukaryotes1. mechanisms of gene regulation in eukaryotesa. DNA wrapped around proteins-to create DNA-Protein complex called chromatinb. Before transcription can begin in eukayotes-the stretch of DNA w. promoter must be released from tight interactions w. proteins-so that RNA Polymerase can contact promoterc. So chromatin remodeling must occur prior to transcription2. Second Level of regulationa. RNA Processing-steps required to produce mature mRNA from primary RNA transcriptb. often alternative splicing patterns occur-diff. combinations of exons unite and make diff. gene products as result3. Third Level of Regulationa. mRNA life span is regulated in eukaryotesb. mRNAs that are active for a long time-are translated more than those w. short life-spanc. All 6 control points are used at certain times in eukaryotic cell4. Chromatin Remodelinga. For arrival of signaling molecule to result in transcription of gene-chromatin around target gene must be remodeledb. DNA is packed inside nucleus so tightly-RNA polymerase cant access it-some of this packing is done by supercoiling5. Chromatin's Basic Structure drawa. most abundant DNA-associated proteins-belong to a group called the histonesb. Chromatin consists of DNA complexed w. histones and proteinsc. Chromatin looked like beads on a string-the beads are called nucleosomesd. X-ray crystallographic data-indicated that each nucleosome consists of DNA wrapped twice around a core of 8 histone proteins-histone H1 seals DNA to each set of 8 nucleosomal histonese. Intimate associaton bet. DNA and histones-occurs in part bec. DNA is neg. charged and histones are pos. chargedf. H1 histones interact w. each other and histones in other nucleosomes-to produce the 30-nanometer fiberg. When chromosomes condense prior to mitosis or meiosis-the scaffold proteins and 30nm fibers are folded and packed into more compact structureh. chromatin structure has profound implications for control of gene expression6. Chromatin Structure is Altered in Active Genesa. the close interaction bet. DNA and histones must be altered-for RNA polykmerase to make contact w. DNAb. Chromatin must be relaxed or decondensed for RNA polymerase to bind to promoter7. Closed DNA Protected from DNAsea. DNase is an enzyme that cuts DNA at random locations-works efficiently only if DNA is in open configurationb. Weintraub and Groudine-compared chromatin structure in genes of blood cells-B-globin and ovalmine genesc. B-globin is protein part of hemoglobin in red blood cells-Ovalbumn is major protein of egg whited. After treating blood cells w. DNase, then analyzing state of the B-globin and obalbumin genes-they found the DNase cut up the B-globin more than the ovalbumin genee. So, chromatin in blood cells was in open configuration at B-globin gene-but closed at ovalbumin gene8. Histone Mutants another type of evidence in support of chromatin remodeling hypothesisa. In mutant yeast cells that dont produce usual complement of histones-genes not normally transcribed are transcribed at high levelsb. the lack of histone proteins prevented assembly of normal chromatinc. Absence of normal histone DNA interactions promotes transcription-so presence of it must prevent transcriptiond. in normal, default state, eukaryotic genes are "off"This is all a new mechanism of negative control9. How is Chromatin Altereda. Chromatin-remodeling=multi-protein machines -which reshape chromatin through series of ATP dependent reactionsb. remodeling complexes contact DNA on surface of nucleosome-twist it in way that allows DNA to loop out from protein core-where it can be transcribedc. Other players in chromatin remodeling add small molcules to histone proteins-8 types of chemical modifications have been discovered=two of best studied processes are acetylation and methylationd. acetylation-associated w. positive control (activation of genes through binding of regulatory protein)-methylation=correlated w. activation of inactivation-depending on which histones are altered10. Histone Transferases (HATs)a. enzymes that modify chromatin-acetylate positively charged lysine residues in histonesb. When a HAT adds acetyle group to selected histones-marked proteins act as binding sites for chromatin remodeling complexes that open DNAc. chromatin is recondensed by histone deacetylases (HDACs)-remove acetyle groups added by HATsd. If HATs are on on switch for transcription HDACs are offState of histone proteins complexed w. DNA is critical determinant of whether transcription occurs11. Chromatin Modification Can Be Inherited--Histone Code Hypothesisa. Histone code-precise patters of chem. modifications of histones contain information-analogous to way the genetic code stores information-the histone code influences whether or a not a particular gene is expressedb. most or some of chemical modifications that distinguish a brain from muscle associated cell-are passed to daughter cell at mitosisc. Histone modifications are example of epigenetic inheritance-a.k.a patters of inheritance not due to diff. in DNA seq.d. Muscle and brain cells differ in part bec. of differently modified histones-not diff. types of genes12. Regulatory Sequences and Regulatory Proteinsa. Once promoter has been exposed to chromatin remodeling-the first step in transcription is interaction w. the TATA-binding proteinb. Other proteins and DNA seq. are involved in controlling gene expression13. Regulatory Sequences that Are Near the Promotera. Regulatory sequences-sections of DNA that are involved-in controlling the activity of genesb. When galactose is absent, S. cerevisiae cells-produce tiny quantities of enzyme req. to metabolize it-when galactose is present transcription of genes encoding the enzyme increasesc. Result: discovery of mutant cells that dont produce any of the 5 enzymes req. for galactose metabolism even if galactose was present SO1. the 5 genes are regulated together2. Normal cells have a CAP-like regulatory protein-that exerts positive control over the 5 genes3. the mutant cells have loss-of-function mutation that disables the regulatory protien14. Promoter-proximal elementsa. regulatory sequences located close to promoter and bind regulatory proteinsb. Have seq. unique to specific genes15. Reg. Sequences Far From Promotera. Antibodies-proteins that bind to specific sites of other molecules-bind to viruses and bacteria and mark them for destructionb. Tonegawa used techniques to place copies of an
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