BIO 344 1st EditionExam # 2 Study Guide Lectures: 9 -16 Lecture 9 (October 1) Chromatin and Gene ExpressionChromatin and Gene Expression- experiment with micrococcal nuclease digestion of DNA showed that something protectsour DNA- chromatin= DNA wrapped in nucleosomeso DNA wrapped around a histone octomer twice- Know histone structural components of the core and H1o Know function of H1o Histones are basic—Arg and Lys are positively charged and neutralize DNA to reduce electrostatic potential and the N-term carries a positive chargeo Allow for negative supercoiling in eukaryotes Understand this process Allows for access to DNA for replicationChromatin- 2 states: euchromatin (accessible) and heterochromatin (inaccessible)o Understand the conformation and implications of eachPromoter Accesso regions must be free of nucleosomeTranscriptional Activators in Chromatin- gal4-VP16= hybrid activator- histone modificationo acetylation by HATs and deacetylation by HDAC—know function and result loosen the grip of histones on DNA or tightens the grip, respectivelyLecture 10 (October 8) Chromatin Remodeling and EpigeneticsRemodeling Complexes- three methods: sliding, acetylation, methylationo know the effects of eachHistone code- different modifications are recognized by proteins that perform different functions/ effect expressiono H3K9me silencing, H3K9ac expression- Protein complexes are readers and writers for the histone codeo Readers recognize residues Bromodomains and chromodomains (know which recognizes what)Activation- transcription factor GCN4 can recruit a coactivator (GCN5, a HAT)o weakens affinity for DNA- Kinase can phosphorylate H3S10 and can recruit TFIIDRepression- Histone methyl transferase (HMT)o Repress or silence transctiption- Histone demethylases (HDM)o Reverse methylationStress Induced epigenetic control of behavior- know Bdnf function and how it causes modification of geneso controlled by multiple promoters—know function of P4o know CHIP techniqueDNA methylation ( different from histone methylation)- DNMT, dMTase, SAM- Happens on CG residueso CpG—cluster in promoter regions and activates those that are nonmethylated DNA methylation propagation blocks gene expression by blocking activator binding to promotersLecture 11 (October 10) SplicingPost Transcriptional regulation of gene expression- splicing, RNA editing, poly adenylationIntrons and Exons- introns are removed, exons code for protein- DNA hybridization—used to visualize introns by mRNAo Understand how this works- Conserved sequenceo Know what occurs at the 5’ and 3’ splicing regions of introns and exonso Branch pointo Consensus= something is recognizing these sites—proteins and RNAs- Splicingo 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 and RNPo know composition (U1,2,4,5,6)o know which snRNPs are bound where and their function- BBP= branch point binding proteino Replaced by U2 to bp with RNA- U2AF recognizes and binds between branch site and 3’ splice siteo Understand branch site “flipping out” Makes 2’ hydroxyl accessible- U1 and U2 basepairing is essential for splicingo Understand compensatory mutations and their effect on splicing- Know why U1 replacement by U6 increases fidelity and how this occurs (helicase—requires energy)- Rearrangements in spliceosome activates splicing- Know difference between intron and exon definitiono Know the effect of mutation in intron or exon definition- S1 mapping: activated cryptic splic siteo S1 nuclease digests ssDNA and ssRNALecture 12 (October 15) Alternative Splicing4 patterns of alternative splicing- optional exon, optional intron, mutually exclusive exclusion, internal splice siteNerve Growth and Sex Determination in Drosophilia- nerve growth and Dscamo splice variants- sex determinationo X chromosome number determines sexo Sxl= sex lethal—starts generating female cells Regulates itself and tra, and tra regulates dsx (double sex)o In males, exon 3 is included in Sxl and excluded in females Including exon 3 includes a stop codon, making the gene nonfunctional Sxl nonfunctional= maleo In tra, exon 2 is included in males and excluded in females Also includes a stop codon, making the gene nonfunctionalo In dsx, females have exon 4 and males have exon 5 and 6 at C terminus- Activators and repressors effect on splicing eventso Repression is more efficient in absence of a regulator- Negative vs. positive regulationo Consider if repressor or activator, then positive or negative and what results will arise from each- Sxl is bound to RNa targeto Repressor competed with U2AF binding—assists U2 snRNP to associate with branch site- Tra with Tra2 regulates Dsx splicingo Tra2 binds on Dsx exon 4o Activator—know binding activity and effect- ESS (exon splicing suppressor)o Know function on SR bindingLecture 13 (October 17) RNA ProcessingPost Transcriptional Regulation of Gene Expression- alternative splicing, capping, poly adenylation- occurs in the nucleus before export into the cytoplasm and translation into protein5’ cap- G added to 5’ triphosphate and G is methylated- Know the function of the cap and the effect of the cap on export- Splicing and exporto cDNA does not export as effectively as spliced WT3’ poly adenylation- GU rich end is cleaved by endonuclease, polyA adds to –OH of CA- 2 factors for cleavageo CPSF and CstFo PAP-- polyA binding protein binds to polyA tail and, with PAP, controls polyA lengtho Know function of poly A tailSplicing, Capping, and poly A for effective export- processing events are coordinated before export—know why- most are co-transcriptionalo know how and why coordination occurs CTD and regulation and order of processingPhosphorylation- CTD—heptad repeat with serines that can be phosphorylatedo Know how this occurso Can block or enhance binding depending on proteins affinityo Serines are targets for kinases (enzymes that add photphates) TFIIHo Mediators—know function Recruitment of RNAP IIo CTD phosphorylation patterns during transcription to control processing Know what phosphorylation allows at each processing event- Histone modifying factors bind to CTDo H3KtriMe- Phosphate is removed and recycled after phophorylationLecture 14 (October 22) RNA EditingRNA editing- editing by ADAR proteinso