BIO 344 1st Edition Lecture 13 Outline of Last Lecture I. Alternative splicing—4 patternsa. Optional exonb. Optional intronc. Mutually exclusive exclusiond. Internal splice siteII. Observing Alternative splicing in nerve growth and sex determination of Drosophiliaa. Nerve growthi. Dscamb. Sex determinationi. Sxl, tra, dsxii. Activators vs. repressorsOutline of Current Lecture I. Post transcriptional regulation of gene expressiona. Alternative splicing, capping, poly adenylationb. Export and translationII. 5’ capa. Why cap—effect on exportIII. 3’ poly A taila. Why adenylation—effect on export and translationIV. Capping, splicing, and poly adenylation for efficient exporta. Coordination of processing eventsV. RecyclingCurrent LecturePost transcriptional regulation of gene expression- Alternative splicing, capping, and poly adenylationo In the nucleuso Then export mRNA into the cytoplasm Translation proteinThese 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.5’ Cap- 5’ ends of mRNA have a triphosphateo 5’ cap= G added to the 5’ triphosphateo 5’ G links to 5’ phosphateo G is methylatedo Added after transcription, NOT part of sequence—added by machinery- Why cap?o Stability Protects from degradationo RNA exporto Translation Ribosome recognizes the cap- Effect of cap on export: experimento DNA template with gene, promoter, RNA polymerase, 32P rNTPs, buffers into a test tubeo +/- (add or not added) enzymes needed for cappingo Harvest frog eggs and microinject into nucleio Separate nucleus and cytoplasmo Run gel, autoradiographyo U6 (involved in splicing)—stays in nucleus so can be used as a marker for the nucleuso In uncapped, bands show clean injection into nucleus, pre-mRNA disappears, mRNA band is smaller than pre-mRNA band (because of splicing), no pre tRNA =all of mRNA is in nucleuso In capped, bands show mRNA mostly in cytoplasm, intron and U6 stay in the nucleus, tRNA stays in the cytoplasm =mRNA in cytoplasm- Caps are important for mRNA export- Is splicing important for RNA export?o cDNA= copy of beta globin copy of DNA that lacks intronso hybridize by FISHo cDNA does not get out as effectively as the WT splicing is important for export3’ poly adenylation- GU rich end is cleaved by endonucleaseo = internal cut- polyA adds to –OH of CAo -----CA-------GU rich o -----CA-OH- o -----CA-AAAAAAAAAAA - The A’s are nontemplated- AAAUAAA—consensus for polyA cleavage- 2 factors for cleavageo CPSF, CstF- polyA polymerase (PAP) added polyA tailo polyA binding protein binds to polyA tail and, with PAP, controls polyA length- important foro stabilityo translationo exporto signals the formation of 3’ mRNA endCapping, Splicing, and polyA for effective export- why are these processing events coordinated before export?o Without coordination export is blockedo Ensures that mRNA was transcribed properlyo Provides a fidelity step for gene expression- Most mRNA processing events are co-transcriptionalo Events happen during transcriptiono Coordinated at C-terminus of RNA Factors at C term domain (CTD) of RNA polymerase II regulate processing- RNAP II is the binding platform for processing machinery CTD is close to mRNA exit as it emerges from the enzyme (5’ end first)- 5’ capping is first, as 5’ end emerges first from enzyme- Then splicing- Then polyA- Phosphorylationo CTD is a heptad repeat with serines that can be phosphorylated Threonine and tyrosine can also be phosphorylatedo –OH group attacks the phosphateo The phosphate adds negative charge and a large functional group Can block or enhance binding depending on a proteins affinity for bindingto phosphate or negative chargeso Serines are targets for kinases (enzymes that add phosphates) TFIIH is a kinase that phosphorylates serine 5o RNAPII is recruited to the activator with the mediator Mediator and RNAP II do not bind well when the CTD is phosphorylated- Drives promoter escape Phosphorylated serine on 5’ end emerges first and capping machinery recognizes thiso CTD phosphorylation patterns change during transcription to control processing Non phosphorylation Serine 5 phosphorylation= 5’ cap Serine 2 and serine 5= splicing Serine 2= poly adenylation- Histone modifying factors bind to CTDo Chromatin modification is connected to transcription in this wayo H3KtriMe is found at active promoters Recruited by serine 5 Early in transcription, close to promoterRecycling- Phosphate needs to be removedo Phosphatases are enzymes that remove the
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