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IUB BIOL-L 211 - Transcription in Eukaryotes II and Transcriptional Regulation in Eukaryotes I

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BIOL-L211 Lecture 22 Outline of Last Lecture I. FACTII. RNA ProcessingIII. PolyadenylationOutline of Current Lecture I. End of PolyadenylationII. Transcriptional TerminationIII. Transcriptional Regulation in EukaryotesIV. Domain Swap ExperimentCurrent LectureTranscription in Eukaryotes II and Transcriptional Regulation in Eukaryotes I(Picking up where we left off)I. End of Polyadenylation:A. PAP (Poly-A Polymerase)1. Binds the 3' end of mRNA strand2. String of adenines on the end of the transcript (around 200)3. This tail helps to prevent degradationB. Poly-A binding proteins1. Stabilizes the Poly-A tail by binding2. Thus increases mRNA stability3. Assists in translation (later)II. Transcriptional TerminationThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.A. Torpedo Model: RNase degradation of smaller piece of RNA triggers termination1. Rat1 associates with Pol IIa. Rat1 is an RNase, which is a type of enzyme that degrades DNAb. In humans, this RNase is known as Xrn22. Poly-A signal is transcribed3. CstF cleaves mRNA4. Rat1 moves from Pol II and is loaded onto mRNA5. Rat1 degrades RNA (5' to 3') until it hits Pol IIa. Degrades uncapped RNAb. Rat1 distinguishes RNA strands by cap vs. no cap6. Pol II dissociates from DNA (transcription is done)B. Allosteric model of Termination (less favored model)1. Pol II transcribes DNA with high processivity2. Processivity decreases after poly-A signal is transcribeda. Poly-A signal causes conformational change in Pol II3. Pol II then spontaneously dissociatesa. Note that CstF still cleaves at the poly-A signal sequenceb. Rat1 still degrades the short pieceIII. Transcriptional Regulation in EukaryotesA. Regulatory Sequences1. Transcription regulators find specific regulatory sequences2. Eukaryotes are more complex than prokaryotesa. Thus more regulatory binding sitesb. Binding sites can be located far upstream or downstream from transcription start siteB. Protein Structure1. Primary Structure: individual bonds between amino acids (sequence)2. Secondary Structure: the way in which small parts of the chain curve and interact with themselvesa. Alpha helicesb. Beta pleated sheets3. Tertiary structure: manner in which different secondary structures interacta. Folded structure of polypeptide chain4. Quaternary structure: large complex of all smaller protein unitsC. Activators: bind DNA and increase transcription1. Domain: Part of a protein structure that has its own structure and is stable apart from the rest of the proteina. Specific binding and activation domains exist for activatorsb. Domains are part of a tertiary structure2. Gal4a. Activator from yeastb. Activates transcription of galactose metabolismc. Controls G AL1 gene (increases transcription 1000-fold)d. Upstream binding sites for Gal4 each bind a dimer of Gal4IV. Domain Swap ExperimentA. Identified the separate DNA-binding and activating regions for Gal4 using transcriptional fusion techniqueB. Experiment 1:1. Transcriptional fusion between core promoter and upstream UAS to lacZa. lacZ was reporter gene2. Transcription of lacZ was measured3. Mutants made of Gal4 lacking each domaina. Wild type Gal4 binds DNA at Gal4 site (UAS) and activates the transcription of the reporter geneb. Gal4 mutant without activating region bound at Gal4 site (UAS) but there was no transcription of the reporter genec. Gal4 mutant without DNA binding domain did not bind DNA at the Gal4site and thus there was not transcription of the reporter geneC. Experiment 2:1. Transcriptional fusion between core promoter and upstream LexA site to lacZ2. Hybrid protein of Gal4 and LexA (a bacterial repressor)a. Wild type LexA bound at LexA site and repressed transcription of lacZb. Fusion protein: bound DNA at the LexA site and activated transcription of lacZD. Showed that Gal4 has two domains, each with separate activating and DNA-binding functionsE. Demonstrated that DNA-binding domains can be swapped between eukaryotes and prokaryotesF. Finally, showed that there similarity in how DNA-binding proteins bind between


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