BIOL-L211 Lecture 20 Outline of Last Lecture I. DNA Binding ProteinsII. The lac operon of E. Coli III. Quorum SensingOutline of Current Lecture I. Major Differences in Transcription in EukaryotesII. Transcriptional Control of a Eukaryotic GeneIII. Transcription Initiation in EukaryotesIV. Elongation in EukaryotesCurrent LectureTranscription in Eukaryotes II. Major Differences in Transcription in EukaryotesA. Eukaryotes lack operons (only prokaryotes have operons)B. DNA compacted by histones (into nucleosomes)C. Prokaryotes lack a nucleus, but in Eukaryotes transcription occurs in the nucleusD. Transcription and translation take place in different areas of the eukaryotic cellE. RNA Polymerase II is the dominant RNA polymeraseF. There are neither sigma factors nor -35/-10 regionsG. Multiple transcription factors are necessaryThese 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.H. Processing of mRNA requires addition of the poly-A tail, splicing to remove introns, and 5' capping of the transcriptII. Transcriptional Control of a Eukaryotic GeneA. Eukaryotic Gene structure:1. Eukaryotes have exons (coding regions) and introns (noncoding regions)B. Promoters and Regulatory Sequences:1. Promoter: Location where general transcription factors bind and recruit RNA polymerase2. Regulatory Sequences: variable sequences that bind regulatory proteinsa. Activators and Repressors = regulatory proteinsb. These sequences can be either upstream or downstream of a promoterc. Typical length: 10s to 100s of kilobases away from core promoterd. Controls whether a gene is turned on or offC. Structure of Core Promoter:1. Set of sequence-specific elements that is required for RNAP to begin transcription2. Important elements: TATA box, BRE, and Inr (Initiator element)a. Note that the initiator is sandwiched at the transcriptional start siteD. RNA Polymerases1. Pol I: Transcribes specialized RNA-encoding genes2. Pol II: Transcribes nearly all protein encoding genesa. Multiple Subunitsb. Catalyzes RNA synthesisc. Claw shape (like in prokaryotes)3. Pol III: Works in conjunction with Pol I4. Pol IV & V: present in plantsIII. Transcription Initiation in EukaryotesA. Three General Steps: Same in eukaryotes and prokaryotes (but different details)1. Binding of RNA Pol II to promoter in closed complex2. Open complex forms (about 13 bp long)3. Initial transcription and promoter escapeB. Preinitiation complex1. Pol II, six general transcription factors, and additional proteins all bind at promoter2. Helps Pol II over transcriptional start site and helps form open complex3. The letters of transcription factors do NOT correspond to event order (order of discovery)a. TFIID: First transcription factor; multi-subunit protein; has TATA Binding Protein (TBP) which binds the TATA box in the core promoterb. TFIIA: Helps TBP (mentioned above) find the TATA boxc. TFIIB: Binds the TFIIB recognition element (BRE) in the core promoterd. TFIIF: Stabilizes protein-protein interactions; brought to Inr element by TFIID, TFIIA, and TFIIBe. TFIIE: Joins complex and recruits TFIIHf. TFIIH: Uses energy from ATP hydrolysis to mediate promoter melting*Remember "H" uses hydrolysis*4. MORE components of the Preinitiation complexa. Mediator Complex: mediates interactions between Pol II and activator proteins and Pol II and general transcription factorsb. Activators: bind enhancers upstream of the promoter to help recruit RNA Pol II to the promoter (like in prokaryotes)C. Abortive Initiation and Promoter Escape1. Promoter Escape Takes two steps2. STEP ONE: Promoter Melting:a. TFIID holds upstream promoter in placeb. TFIIH binds downstream of RNAPc. TFIIH uses ATP hydrolysis and feeds dsDNA into polymerase and separates the strands3. Abortive Initiation occurs before the second step in promoter escapea. Pol II stalls at the promoter, synthesizing numerous short transcripts4. STEP TWO: Phosphorylation of Pol IIa. Pol II has a tail (carboxyl-terminated domain [CTD])b. Tail comes to promoter in an unphosphorylated statec. TFIIH phosphorylates the tail to help Pol II escape to elongationIV. Elongation in EukaryotesA. Elongation begins post promoter escapeB. Elongation follows similar mechanism to prokaryotes (same RNA nucleotides, same chemical reaction for RNA synthesis)C. Elongation Factors: stimulate transcript elongation1. Some help displace initiation factors2. Others stimulate Pol II proofreading activityD. Histones cause problems:1. Physical roadblocks in the form of nucleosomes that impede
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