Chapter29Powerpoint 11.45.23 PM

Chapter29Powerpoint 11.45.23 PM - CHAPTER 29: RNA SYNTHESIS...

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Unformatted text preview: CHAPTER 29: RNA SYNTHESIS (Transcription) AND SPLICING Transcription: DNA RNA LECTURE TOPICS • • • Prokaryotic transcription • Post-transcriptional RNA modifications (theme for all topics) • RNA splicing mechanisms Eukaryotic transcription Transcription factors (DNA/protein and protein/protein interactions) CHAPTER 29: RNA SYNTHESIS (Transciption) AND SPLICING Transciption: RNA DNA TO REVIEW: Chapter 4 Notes Review Ch.4 notes Transcription: DNA RNA Reaction is: (n> or = 0; i.e., NO primer needed!) [DNA template] + (NMP)n + NTP (NMP)n+1 + PPi STAGES: • • • Initiation [No primer needed!] Elongation Termination MECHANISM: Same as DNA polymerase • 3’-OH attack on α-P of next (r)NTP 2Pi ++) 2 Asp, 2 metal (Mg2 ions play a role in 5’ to 3’ RNA polymerase mechanism [similar to DNA Pol I active site] 3` OH α-P ++) 2 Asp, 2 metal (Mg2 ions in Pol I active site play a role in 5’ to 3’ polymerase mechanism [See Ch.28 notes] d 3` OH α-P E. Coli RNA Polymerase subunits core Sigma (σ): used for initiation only (α2, β, β’) core σ holoenzyme E. Coli RNA Polymerase: Roles of subunits E. Coli RNA Polymerase (holoenzyme) on T7 phage DNA Omit NTPs and RNA polymerase sticks to “Promoter sites” Question: Is this a sequence specific Protein/DNA interaction? Promoter Sequences: Identify by DNA Foot Printing Promoter DNA Identify by Gel Electrophoresis Promoter Sequences: Identify by DNA Foot Printing RNA Polymerase - + Missing cuts Prokaryotic Start Signals (Promoter) coding strand sequences •Promoters of different gene classes have variations of the 2 conserved consensus sequences. • All have different sigma factors for transcription initiation Prokaryotic Promoter: σ subunit -10 sequence recognition σ subunit has α-helix for -10 sequence recognition Most frequent -10 bases Prokaryotic RNA polymerase holoenzyme complex: σ subunit contacts -10 -10 and -35 sequence elements RNA Polymerase holoenzyme unwinds DNA (At promoter) Transcription Reaction X = Purine (G/A) First base (5`) G/A 5` 3` Elongation G/A growing RNA TRANSCRIPTION BUBBLE: Elongation 100% processive OH [A helix ~ 12 bp] Rate : 50 bases/sec TRANSCRIPTION BUBBLE (Model based on enzyme structure): Elongation Template strand A structure in the polymerase forces separation of new RNA from the RNA-DNA hybrid [A DNA-A helix] Termination of Transcription 1) Rich in GC base pairs RNA transcript 2) Run of U’s complementary DNA is: GC GC ● CG CG Rho(ρ) protein dependent termination ●●● (+) ρ (-) ρ ρ added at different times Mechanism of Rho protein dependent termination 3’ (hexamer) The “Central Dogma” of molecular biology (Ch.5, p21) 10 -4,-5 10-8 DNA transcription Replication DNA Repair improves to 10-9 10-3, -4 RNA Reverse transcription translation PROTEIN No error correcting! 10-4 FEATURES OF PROCESSES Accuracy - RELATIVE Signals – STARTS AND STOPS Stages – INITIATION, ELONGATION, TERMINATION E. Coli RNA Polymerase INITIATION Holoenzyme: 450kd, 4 subunits Sigma subunit: promoter recognition ELONGATION Core enzyme: RNA chain elongation Contains catalytic site is 100% processive Functions of RNA Polymerase Searches for initiation site: ~2000 promoters Unwinds DNA template Select correct NTP for: base-pairing for initiation and elongation in RNA synthesis Detect termination signals Interact with activator or repressor proteins: controls transcriptional levels Needs no primer to initiate [A or G is 5’-end] No error correcting activity: Error rate is 10 –4 to-5 Post-Transcriptional Processing of RNA Examples • • rRNA precursors Modified bases E. Coli rRNA transcript: Processed to create mature rRNAs and tRNA Many cuts by several ribonucleases Processing is post-transcriptional • Endo- and exonucleases required Post-transcriptional base changes: • Bases and ribose are chemically modified • Many unusual bases are found in tRNA Unusual bases in tRNA 5 [rT] 1 5 [ψ] Inhibitors of Transcription Examples • • Rifampicin Actinomcycin D • • prokaryotesd- and eukaryotes α-amanitin • eukaryotes Rifampicin: Binds to RNA Polymerase holoenzyme where DNA-RNA hybrid starts (after 2-3 bases connected) • Competes with DNA-RNA hybrid • Inhibits phosphodiester bond formation Rifamycin: Initiation (binds to $ subunit and prevents P- diester bond formation) Looks like a base pair Actinomycin D: Elongation inhibitor Actinomycin D *intercalates between DNA base pairs 5’ G 3’ C Actinomycin D G C 3’ RNA Elongation Inhibitor Stops RNA Polymerase Cancer Drug 5’ * Intercalates (inserts): like acridine orange TRANSCRIPTION AND TRANSLATION: Prokaryotic vs Eukaryotic COUPLED SEPARATE COMPARTMENTS Prokaryotic and Eukaryotic RNA Polymerases: Conserved structure of largest subunits Thermus aquaticus yeast CHAPTER 29: RNA SYNTHESIS (Transcription) AND SPLICING Transcription: DNA RNA LECTURE TOPICS • • • Procaryotic transcription • Post-transcriptional RNA modifications (theme for all topics) • RNA splicing mechanisms Eucaryotic transcription Transcription factors (DNA/protein and protein/protein interactions) Mushrooms and Eukaryotic Transcription Amanita muscaria: fly agaric [Poisonous, hallucinogenic, common] Amanita virosa: Destroying angel [Poisonous, deadly, common] Mushrooms and Eukaryotic Transcription Amanita phalloides: Death Cap [Poisonous, deadly, common] α-amanitin: eukaryotic RNA polymerase II inhibitor 3 Eukaryotic nuclear RNA polymerases: many subunits α-amanitin: insensitive α-amanitin: Kd = 10-6 M α-amanitin: Kd = 10-9 M Transcription Promoters: Prokaryotic vs Eukaryotic Consensus RNA polymerase recognition sequences Promoter sequences for RNA polymerase II recognition Can be either strand +1 Most frequent base [consensus sequence] Second most frequent base [DNA sense (coding) strand] Some promoter recognition sequences for eukaryotic RNA polymerases I-III +1 +1 UPE - Upstream promoter element +1 rlnr - ribosomal RNA initiator element (RNA Pol I) Inr - initiator element (RNA Pol II) near transcription start DPE - Downstream promoter element +1 +1 CHAPTER 29: RNA SYNTHESIS (Transcription) AND SPLICING Transcription: DNA RNA LECTURE TOPICS • Transcription factors bind to promoter recognition sequences, mediated by: • DNA/protein and protein/protein interactions Transcription factors lurk in the shadows TATA-box binding protein RNA Polymerase II TATA box Transcription factors (proteins): (TBP) TATA-box binding protein [a subunit of TFIID] TATA box minor groove TBP (30 kD) is the DNA recognition subunit of TFIID (700 kD) TATA-box binding protein TATA box major groove • • • • • • Binds to minor groove Bends DNA 80-90o Opens minor groove Very asymmetric interaction Sequence specific Intercalates between 4 Phe`s and minor groove bases Initiation: Transcription Factor (TF) Complex Assembly (30 kD subunit of TFIID) IIA binds to IID and to DNA Transcription initiation complex formation +D +A +B *Stepwise addition of TF’s BEFORE start of transcription +F,E,H +Pol II Initiation of transcription [F is a helicase] new RNA More Transcription Factors that bind to specific sequences: When bound to DNA, these proteins interact with other TF’s and/or RNA Pol II subunits to stimulate transcription of specific classes of genes Sp1 protein HSTF (C) CAAT-Box Binding Protein also Enhancer/activating protein complex interacts with promoters from a distance: (Result in efficient initiation and enhanced transcription) 5` Activator protein F Promoter 3` Glucocorticoid Hormone Enhancer System +1 1 Receptor 2 3 Binding to RNA Polymerase and transcription initiation Summary Transcription Factor/Initiation Complexes ARE Complex!!! Post-Transcriptional Processing of Eukaryotic RNA Examples • • • rRNA precursors tRNA precursors Modified bases Precursor tRNA processing (Yeast tRNATyr) Enzyme adds CCA Modified bases Arrows point to nuclease cut sites Splice site Eukaryotic rRNA transcripts is a large pre-rRNA that is processed to create 3 mature rRNAs. Processing includes • Endonucleases • Exonucleases • Base modifications Eukaryotic mRNA : Post transcriptional modifications • Splicing (delete introns, connect exons) • 5`- cap added • 3` - poly (A) added • RNA editing Protein coding Sequence 5` cap AUG--------------UAG Start Stop AAAA—A-OH 3` 5` cap on RNA Polymerase II transcripts (mRNA) GTP G PPi 3 types of caps (0,1,2) Pi one or both Cap Transcription Termination and Poly(A) addition * Cut * Up to 250 A’s added RNA Editing of apolipoprotein B-100 (In Liver) Amino acids [CAA = Gln] CU [UAA = Stop] (In small intestine) Amino acids CHAPTER 29: RNA SYNTHESIS (Transcription) AND SPLICING Transcription: DNA RNA LECTURE TOPICS RNA splicing mechanisms RNA SPLICING Eukaryotic mRNA Intron Splicing: 3 Conserved sequence signals [20-50 bases] 5` 3` 5` upstream downstream [Definitions] 3` A splicing mutation in the β-globin gene causes thalassemia [Mutation creates a new 3` splice site.] 1st GU not used New intron is spliced out Truncated protein Truncated protein is RNA Splicing Mechanisms: Transesterification reactions • Conserve phosphodiester bonds • [make one / break one] (δ+) 2 .. (δ–) Reaction Mechanism: • Nucleophilic attack on Phosphate by δ– charge of unpaired electrons of ribose OH- group. • Exchange R2 and R3. RNA Splicing Mechanisms: Transesterification reactions Conserve phosphodiester bonds [break one - make one] 2 Reaction Mechanism: Nucleophilic attack by δ– charge of unpaired electrons of ribose OH- group. Intron Splicing Mechanism : 2 transesterifications 2 1 δ- δ+ - + δ- δ+ * - + (mRNA) To 3` of intron Branch Point (A): 2`, 3`, 5`- (A) phosphotriester 5` G of intron Next Base 5’ to 3’ SPLICEOSOMES: Splicing in the Nucleus Fluorescent spliceosome proteins There are about 50 spliceosome proteins Nucleus Spliceosome components (snRPs) Small Nuclear Ribonucleoprotein Particles: Have RNA and proteins and are involved in splicing (defects in SLE – lupus) One Role of snRNP RNAs: [example] U1 RNA base pairs with 5`-Splice site SPLICEOSOMES : ORDERED ASSEMBLY SnRPs [SnRNP Complex] SPLICEOSOMES: ORDERED ASSEMBLY - U1 and U4 SPLICEOSOMES: Transesterifications and splicing products (1) (1) (2) (2) Spliceosome catalytic center U6 and U2 hold everything else in place U5 snRNA Branch site U1 snRNA U1 and U5 hold exons in place Coupling transcription to pre-mRNA processing: 1. 2. 3. 5’ capping Splicing Poly (A) addition TFIIH phosphorylates the CTD (Carboxy terminal domain of Pol II. 1. signals switch from initiation to elongation 2. CTD binds factors needed for capping, splicing, and adding poly(A). * RIBOZYME: Self- Splicing RNA (Ex: Tetrahymena rRNA precursor) (rRNA) fold + cut Intron is still a ribozyme Base-paired and folded RNA has special sites for: G-binding guide Sequence fold + cut fold + cut fold + cut RIBOZYME structure: Tetrahymena rRNA precursor. HAMMERHEAD RIBOZYME activities can be engineered EVOLUTION OF SPLICING RIBOZYMES: RNA alone RNA + Proteins involved Same mechanisms, lariat introns ALTERNATIVE SPLICING: Different mRNAs from same pre-mRNA See also immunoglobulin example in Chapter 4 EVOLUTION: Started with an RNA world? RNA enzyme (ribozyme) Protein DNA Key Points : Eukaryotic mRNA splicing • • • • • • • • 3 Sequence Signals 2 Transesterifications (break and make 5`-P-3` bonds) “Spliceosomes” in nucleus snRNPs - RNA and Protein snRNAs base pair with pre-mRNA Ordered assembly of spliceosomes Ribozymes – Self splicing RNA Evolution? Splicing (+/-) Proteins ...
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