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Outline10Lectures17-19 - BIS101/Engebrecht S10...

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Unformatted text preview: BIS101/Engebrecht S10 Outline10Lectures17-19 Regulation of Gene Expression in Prokaryotes and Eukaryotes Reading in 8th edition Chapter 10, 301-316; 9th edition Chapter 10, 351-379 I. The lac operon of E. coli An operon = a genetic unit of coordinately expressed genes that are transcribed on a single polycistronic mRNA. The genes within the lac operon encode proteins responsible for the metabolism of the sugar lactose as well as the regulatory elements to control their precise expression. The genetic analysis of this system by Jacob and Monod provided the first understanding of how genes were regulated at the transcriptional level. (Please see Handout16 on “Possible models of transcriptional activation”) a. Structure of the lac operon, function of encoded proteins (lacP = promoter for all genes of the lac operon; lacO = operator, regulatory region and binding site for lacI for allgenes in the lac operon; lacZ = encodes betagalactosidase that converts lactose into a form that isusable for the cell and produces the inducer allo-lactose; lacy = encodes the lactose permease that brings lactose into the cell; lacA = encodes lactose transacetylase, function unclear, we will not discuss it further), the roles of lactose and allolactose. Induction = relief of repression; allolactose is a derivative of lactose that inactivates the repressor and leads to expression of the lac gene = inducer. b. Lac operon characteristics: 1000-fold up-regulation, quick down regulation. c. LacI repressor protein (repressor = blocks expression of genes) and its regulatory target sequence the lacO operator (operator = the site on the DNA to which the repressor binds). d. Basic mechanisms of induction and repression of the lac operon, LacI repressor, promoter function and operator function. e. Mutational analysis that established the operon model. (Please see Handout17 on Merodiploid analysis) i. Mutations in lacI (constitutive and non-inducible repressors) ii. Mutations in lacO (constitutive operators) iii. Cis/trans test in merodiploids. LacI mutations 1) lacIc = constitutive expression mechanism: no operator binding; therefore, mutations define DNA binding site 2) lacIns or lacIs = non-inducible expression, superrepressor mechanism: no allolactose binding; therefore, mutations define inducer binding site 3) lacIQ, lacISQ = less induction mechanism: promoter mutation of the lacI gene leading to higher repressor levels. f. Mutational analysis to define structure-function relationship in a single protein: the lacI repressor i. DNA binding-site ii. Allo-lactose binding-site iii. Tetramer formation g. Catabolite repression = catabolic breakdown of glucose (a preferred carbon source for E. coli) prevents the expression of the lac operon by lactose. i. catabolite activator protein (CAP) ii. cyclic AMP iii. DNA bending and RNA polymerase recruitment II. Phage lambda: A paradigm for a regulatory switch The switch between lysogeny (phage genome is integrated into the bacterial chromosome and silent) and lytic cycle (active replication and transcription of the phage genome) in phage lambda is regulated by a transcriptional switch that serves as a paradigm how different developmental fates can be regulated. Prokaryotes vs. Eukaryotes: The paradigms of gene regulation developed in bacteria were positive and negative transcriptional control by sequence-specific DNA binding proteins that underwent a conformational change upon binding to a small molecule. Short half-lives of RNAs and proteins ensures a quick response of the steady-state levels. The basic molecular mechanism of transcription in eukaryotes is similar to that in prokaryotes. However, additional complexity in gene and chromosome structure, RNA metabolism, transcriptional regulation, RNA and protein transport, localization and stability, posttranscriptional and post-translational modification allow for far more regulatory opportunities in complex organisms. This is due in part to the difference in cellular organization between prokaryotes and eukaryotes. Please see Handout18 “Eukaryotes vs. Prokaryotes” – this emphasizes the differences between eukaryotes and prokaryotes with respect to regulation of gene expression at the transcription, post-transcriptional and translational level. Also take a look at the Handout19 “Prokaryotic and Eukaryotic Genes”, which shows in cartoon the key features of genes and their regulatory regions. Reading in 8th edition Chapter 10, 316-334; 9th edition Chapter 11, 385-411 (For additional information see 8th edition Chapter 18, 575-606) III. Control of Eukaryotic Gene Expression at the DNA level-more complex chromosome organization influences gene expression a. Chromatin structure (euchromatin = active regions of the genome where genes are expressed; heterochromatin = regions of the genome which are not expressed due to compaction) b. Histone modification “the histone code”-methylation, acetylation, phosphorylation c. DNA methylation (CpG methylation) and epigenetic inheritance IV. Regulation at the transcriptional level- more complex transcriptional apparatus (Chapter 11, pg. 350-361) a. Three RNA polymerases: RNA polymerase I = transcribes ribosomal RNA; RNA polymerase II = transcribes mRNA (to make protein) and RNA polymerase III = transcribes tRNA, 5S RNA (a component of the ribosome), and small RNAs b. Combinatorial control of transcription by transcription factors and their regulatory DNA binding site: enhancers = cis-acting sequences that can greatly increase transciription rates from promoters on the same DNA molecule; silencers = cis-acting sequences that are bound by repressors, thereby inhibiting activators and reducing transcription. c. Hormone-regulated transcription factors V. Regulation at the RNA level (Chapter 10, pg. 306-311) a. More complex mRNA structure i. cap = 7-methylguanosine residue linked to 5’ end of transcript ii. exons = DNA segments that code for the structure of the protein; introns = intervening sequences that interrupt the coding sequences. iii. PolyA tail = stretches of adenosine residues added at the 3’ ends of mRNA b. Splicing = removal of introns – Please see Handout20 on “Splicing” c. RNA transport d. RNA stability VI. Regulation at the protein level a. Translation control b. Protein sorting/transport c. Protein modification (phosphorylation, glycosylation) d. Regulated protein turnover (ubiquitin) ----------------------------------------------------------------------------------------------------------I am giving you the information below to help you keep track of the large number of proteins that function in eukaryotic transcription. I do not expect you to memorize these, but wanted to provide you with a guide to help in our discussion. RNA polymerase II holoenzyme: This is the eukaryotic polymerase that transcribes mRNAs. It is a very large, multi-protein complex. TBP = TATA binding protein: This a general transcription factor that binds to the TATA sequence in the promoter of genes encoding for mRNAs. TAFs = TBP associated factors: These are general transcription factors that bind TBP. TBP and TAFs make up TFIID. TFIIB, TFIID, TFIIE, TFIIF, TFIIH = Transcription factors for RNA polymerase II: These are large protein complexes that associate with the RNA polymerase II holoenzyme and are required for basal transcription. Each of the 5 transcription factor complexes are made up of 10 or more subunits. These complexes were identified by biochemical purification and their letter denotes the fraction that they were found in. Mutations in any of the above general transcription machinery would perturb basal transcription from all genes encoding mRNAs. (There are also transcription factor complexes for RNA polymerase I and III. These are also designated by TFIA, TFIB (for pol I) and TFIIIA, TFIIIB (for pol III). Some transcription factors are general and shared between all 3 polymerases, others are specific for a given RNA polymerase). Activators: These bind enhancer sequences in a DNA sequence specific manner and stimulate transcription. Co-activators: Bring activators and the basic transcriptional machinery in close proximity. One example of a co-activator is histone acetylase, which alter the compaction state of chromatin. Repressors: The proteins bind silencer sequences in a DNA sequence specific manner and repress transcription. Co-repressors: Bring repressors and the basic transcriptional machinery in close proximity. Mutations in these regulated transcription factors would result in a change in the level of regulated transcription of those genes encoding mRNAs that have binding sites for the factors. ...
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