Bio 115 TA Study Guide for Midterm 2

Bio 115 TA Study Guide for Midterm 2 - Patrick Yuh Winter...

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Unformatted text preview: Patrick Yuh Winter 2011 Bio 115 Study Guide for Midterm 2 (bold = important) Reminder of major topics 1. Transcriptional activation activators, Mediator, ‘big picture’ of txn 2. Chromatin nucleosomes, histone modification 3. RNA processing, structure, function 5’ capping, splicing, polyadenylation 4. Splicing mechanism, spliceosome, alternative splicing Terms: basal transcription RNA world hypothesis histones activated transcription pre‐mRNA splicing (know histone tails transcriptional activator splicing anything, if you 10 nm ‘beads on a string’ know what’s good for inducible vs. constitutive txn 30 nm fiber you; see below for more) MNase Gal4 5’ and 3’ exons linker DNA LexA intron histone octamer sequence specificity 5’ and 3’ splice sites supercoiling activation domains branchpoint A packing ratio DNA binding domains lariat higher order chromatin Zn fingers spliceosome architecture helix‐turn‐helix 2 chemical steps of splicing DNase I hypersensitivity Leu zippers poly A signal types of histone modific’ns homeodomains RNase P histone code hypothesis squelching covariance Swi/Snf Mediator compensatory mutation octamer sliding combinatorial control of HIV Tat protein & TAR elem. octamer disassembly gene expression alternative splicing 5’ capping chromatin exon inclusion polyadenylation repressed transcription exon skipping ribozymes nucleosome 1 Concepts: o dimerization of DNA binding proteins o binding cooperativity o the effect of nucleosomes on transcription (slides 213‐215, 217) o what happens when you get rid of nucleosomes (slide 216) 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 pre‐mRNA processing? o pre‐mRNA processing occurs co‐transcriptionally → 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 pre‐mRNA 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’) 2 Techniques to add to your toolbox: Yeast two‐hybrid 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: a super‐limited DNase I digest; it will cleave wherever the chromatin is accessible, i.e. a longer stretch of DNA that is nucleosome free these DNA sequences are referred to as hypersensitive (HS) sites actively transcribed genes will contain at least one or two HS sites inactive genes do not contain any HS sites Indirect end labeling MNase or DNase I digest → nucleosome‐sized DNA fragments restriction digest on purified DNA fragments, Southern blot → one nucleosome ladder if in phase (slide 221 gel B), many nucleosome ladders (that look like a random ladder/smear) if out of phase (slide 221 gel A) Immunofluorescence staining (slides 231, 237, 238) like a Western blot, except you want to see where your target protein is localized in the cell add 1° antibodies to fixed cells; add 2° antibodies that are conjugated to fluorescent tags In vitro binding assay (slide 260) express a GST‐fusion protein and couple it to beads → make a column with these beads pass cell extract over the column see what binds your protein Analysis of functional RNAs (slides 278, 282) is RNA function due to sequence or 2° structure? if structure, must see a new phenotype when structure is lost, AND rescue of phenotype when compensatory mutation is made In vitro splicing reaction use nuclear extract from a cell line to do splicing in vitro, using a radiolabeled pre‐mRNA substrate (transcribed in vitro) learn how to ‘read’ a splicing gel 3 ...
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