BIO 344 1st Edition Lecture 11 Outline of Last Lecture I. Remodeling ComplexesII. Histone codea. Reading and writing the codeIII. ActivationIV. RepressionV. Dynamica. PropogationVI. Stress induced epigenetic control of behaviora. Bdnfb. Exons and promotersVII. DNA methylationOutline of Current Lecture I. Post transcriptional regulation of gene expressionII. Introns and exonsa. DNA hybridizationb. Conserved sequencesIII. Splicing stagesa. Assembly of spliceosomeThese 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.i. snRNPIV. intron vs. econ definitiona. mutations in exon vs intron definition Current LectureSplicing- post transcriptional regulation of gene expressiono splicingo RNA editingo Poly adenylationIntrons and Exons- Intron= sequence removed from pre-RNA by RNA processing enzymes- DNA transcriptpre-mRNARNA splices introns ligates exonsmature mRNA- DNA hybridization= used to visualize introns by mRNAo Take pre-mRNA, hybridize cDNA to it—will bind in certain regions, those not bound make loops, and the loops are the intronso Shows that pre mRNA does not exactly correspond to mRNA- Conserved Sequenceo Comparison of exon/intron junctionso Line up splice sites 5’ splicing region at end of exon (exon-intron junction) is spliced and 3’ splicing region at beginning of next exon (intron-exon junction) to bind the exons and make mature mRNA Splicing regions—are these consensus sequences for splicing at the junctions?- GU at 5’ splice site of intron and AG at 3’ splice siteo Not unique enough for recognitiono Also an A at the branch point- Consensus sequence indicates that something needs to recognize these sites. What recognizes them?o Proteins and RNAso Example: Beta Thalassemias See slide for experiment details- B0= no beta globin present- B+= low level of structurally normal beta globin foundo G to A mutation in intron 1 Affects the splice site by splicing in upstream position that causes the intron to be included and changes the reading frame Causes extra amino acids to be included (from the intron sequence being included), incorrect amino acids past the mutation site (due to altered reading frame), and early termination- HbA= adult hemoglobin- deltaB= no adult hemoglobin but still express embryonic hemoglobin- Splicing o the 2’ hydroxyl of A branch site is the nucleophile that attacks the 5’ splice site o 3’ hydroxyl of 5’ splice site attacks 3’ splice siteo 3’ hydroxyl ligation siteAssembly of spliceosome- snRNP= small nuclear ribonucleopreotein particles- RNP= RNA and protein compelexo RNA is composed of small nuclear RNAs U1,U2,U4,U5,U6o 5’ splice site bound to U1 snRNPo BBP= branch point binding protein—is replaces by U2 to base pair with RNAo U2AF recognizes and binds between branch site and 3’ splice siteo Branch point adenosine doesn’t bind to U2, so branch point “flips out” U2 base pairs with RNa at expense of evicting adenosine to present the 2’hydroxyl for attack If not, 2’ hydroxyl would not be accessibleo Base pair of U1 to 5’ and U2 base pairs to the 3’ end Essential for splicing if mRNA is mutated to prevent base pairing, splicing is prevented If compensatory mutation (mutation on one and a complementary mutation on the other) and can get function back, shows that base pairing is essential for functiono U1 is replaced by u6 snRNA RNA helicase drives this and other critical rearrangements- Requires energy—ATP ADP Increases fidelity two things used to recognize the sequence, two checks of site identity Branch point and U2 do similar rearrangemento Rearrangements in spliceosome activates splicing Brings 5’ 3’ splice sites together- 5’ exon attacks intron (U5)Intron versus Exon Definition- in different organisms, exons may be a set size and introns may vary in size or introns area set size and exons vary in size- intron definition= U1 and U2 recruit spliceosome communication across intron- exon definition= U1 and U2 communicate across exon so intron can evolve to any length o SR proteins bind to exons and link snRNPs- Effect of mutations in exon vs. intron definitiono Exon definition mutation Causes skipping an exono Intron definition mutation Intron can be included because skipped a splicing event Can activate cryptic splice site (altered splicing)- Mutation in cryptic splice site to make a novel splice siteo S1 mapping S1 nuclease digests ssDNA and ssRNA Beta thal patient has longer transcript due to inclusion activated cyrpticsplice
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