Bio 115 Study Guide 2

Bio 115 Study Guide 2 - Patrick Yuh 18 Feb 2009 Bio 115...

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Unformatted text preview: Patrick Yuh 18 Feb 2009 Bio 115 Study Guide for Midterm 2 (bold = important) Reminder of major topics 1. Transcriptional activation 2. Chromatin 3. PremRNA processing (5' capping, polyadenylation, splicing) 4. Alternative splicing, tRNA & rRNA processing 5. mRNA decay, miRNAs, siRNAs (RNAi) Terms: basal transcription MNase activated transcription core particle repressed transcription linker DNA inducible vs. constitutive txn histone octamer Gal4 supercoiling LexA packing ratio sequence specificity higher order chromatin DNA binding domains architecture Zn fingers DNase I hypersensitivity helixturnhelix types of histone modific'ns Leu zippers HATs basic helixloophelix HDACs (bHLH) histone code hypothesis squelching Swi/Snf complex Mediator octamer sliding TAFs octamer disassembly combinatorial control of 5' capping gene expression polyadenylation chromatin premRNA splicing (know heterochromatin splicing anything, if you know what's good for you; euchromatin see below for more) nucleosome 5' and 3' exons histones intron histone tails 5' and 3' splice sites 10 nm `beads on a string' branchpoint A 30 nm fiber polypyrimidine tract (PYT) lariat ribozymes RNA world hypothesis spliceosome 2 chemical steps of splicing poly A signal RNase P group I & II introns covariance compensatory mutation HIV Tat protein & TAR elem. alternative splicing exon inclusion exon skipping mRNA decay mRNA halflife microRNA siRNA RNAi knockdown transferrin receptor iron response element AUrich element Drosha Dicer sense & antisense RNA Concepts: o dimerization of DNA binding proteins o binding cooperativity o the effect of nucleosomes on transcription (slides 237239, 241) o what happens when you get rid of nucleosomes (slide 240) o in the context of chromatin, there is now a 3rd transcriptional state (another layer in the regulation layer cake) o chromatin remodeling affects and can regulate transcription o what is the point of premRNA processing? o premRNA processing occurs cotranscriptionally what effects does this have? o understand the details of splicing what bonds are broken and made, and where the intermediates and final products of splicing what are spliceosomes made of? how the spliceosome recognizes and binds to the premRNA spliceosome assembly who's on first? second? third? does splicing require energy input? if yes, for what part? if no, why not? how did splicing evolve? o alternative splicing and what makes a splice site strong or weak (`good' or `bad') o rRNA processing and modification o tRNA processing and modification (recall RNase P from earlier) o regulation of gene expression by mRNA turnover o regulation of gene expression by expression of miRNAs and siRNAs 2 Techniques to add to your toolbox: Yeast twohybrid assay you need three ingredients what are they? draw them out and walk through the process of setting up a Y2H be familiar with these terms: bait, prey, reporter, activation, DBD, AD Chromatin immunoprecipitation (ChIP) ChIP is like `in vivo footprinting' what does a ChIP tell you? what is the readout? Micrococcal nuclease digest (MNase) what's the difference between MNase and DNase? what do you get with a limited vs. complete digest? DNase I hypersensitivity the idea: DNase I will cleave wherever the chromatin is accessible, i.e. in specific places where nucleosomes are not present these DNA sequences are referred to as hypersensitive (HS) sites actively transcribed genes will contain at least one HS site in the promoter inactive genes do not contain any HS sites Indirect end labeling MNase or DNase I digest nucleosome ladder restriction digest on nucleosome particles, Southern blot one nucleosome ladder if in phase (slide 243 gel B), many nucleosome ladders (that look like a random ladder/smear) if out of phase (slide 243 gel A) Immunofluorescence staining (slide 260) like a Western blot, except you want to see where your target protein is localized in the cell add antibodies to fixed cells; antibodies are conjugated to fluorescent tags In vitro binding assay (slide 277) express a GSTfusion protein and couple it to beads make a column with these beads pass cell extract over the column see what binds your protein In vitro splicing reaction use nuclear extract from a cell line to do splicing in vitro, using a radiolabeled premRNA substrate (transcribed in vitro) learn how to `read' a splicing gel RTPCR (reverse transcriptasePCR; slide 328) use RNA as template make DNA copies 3 ...
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This document was uploaded on 08/12/2009.

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