BGYB10&B11 Final Exam Study Guide

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University of Toronto Scarborough
Biological Sciences
Shelley A.Brunt

BGY10 J Final #1 Lecture #6: Nucleus J Chapter 12 - nucleus = site of storage for genetic material to be replicated/expressed - nuclear envelope surrounds nucleus J double membrane barrier - cell in interphase has: o chromatin = chromosome as highly extended nucleoprotein fibers o nuclear matrix = fibrillar network w/ protein J residual filamentous Protienaceous matrix o nucleoli = 1+, electron-dense structure J site of rRNA synthesis and ribosome assembly o nucleoplasm = fluid solutes dissolved in - proteins, RNA, ribonucleoprotein particles (RNP) have to move in/out of nucleus - fusion of 2 membranes form pore = complex assemblyof protein J 3000 pores/cell J nuclear pore complex = gateway to nucleus o RNAs and rb subunits out o proteins in o snRNAs both ways o RNP particles assembled in cytoplasm - often OM studded w/ rb and attached to ER - nuclear lamina = dense meshwork (made up of lamins = intermediate filaments), lies on inner surface of nuclear envelope, disintegrates during mitosis and results in phosphorylation J mechanical support for envelopes and site of attachment of chromatin - nucleus has major control J e.g. HeLa cells: contain 10,000,000 ribosomes o single nucleus has to import 560,000 proteins and import 14,000 rb subunits every minute - nuclear pore complex (NPC) = fills pore like a stopper o a larger supramolecular complex J 30x mass of ribosome o octagonal symmetry o depending on org, can have 30-50 diff proteins = nucleoproins (nups) o large materials (e.g. gold particles and rb subunits) can penetrate nuclear pores but is not an open channel o has spoke-ring assembly, nuclear basket, central plug (transporter), 8 cytoplasmic filaments - proteins that enter nucleus have nuclear localization signal J stretches of positively charged AA J function as ZZZ[ - proteins that move out have nuclear export signals - proteins (karyopherins) that function as transport receptors moving macromolecules across nuclear envelopes J importins: move macromolecules in, exportins: move macromolecules out - importing proteins from cytoplasm into nucleus J GTP bring protein (high in nucleus, low in cytoplasm) J conformational change of transporter (open channel) o Z}]K9]K}]LHlJJ binds to protein/cargo to be transported o accessory RCC1 in nucleus converts Ran-GDP to Ran-GTP, maintaining gradient o ]K}]LJK}Z l]L} }oZK Z}ZL-GTP o release w/ hydrolysis J needs accessory Ran-GAP1 - RNA usually move thru NPC as ribonucleoprotein complex (RNP) (except tRNA J recognized directly by exportin-t) o RNP carries nuclear export signal J recognized by exportins o mRNAsmove in complex w/ hnRNP-exportin and Ran-GTP and must be mature mRNA for export - genome has to be packed in nucleus to fit o human cell: 6 billion bp divided w/in 46 chromosomes (diploid) J each unreplicated chromosome contains single continuous DNA molecule (ds DNA) o if each bp 0.34nm in length, 6 billion bp = 2 meters long o each bp binds 5 water molecules J expands volume - metaphase chromosome = X shaped kind o centromere = has AT rich DNA (satellite) randomly repeated (constitutive heterochromatin) J have microtubule attachment site for separation of chromosome in cell division o telomere = (tips of chromatid) contains repeated sequence, for chromosome replication, protects chromosome from nuclease, has telomerase activity to maintain chromosome length in DNA rep/meiosis - only about 5% of human DNA encodes functional RNAsand proteins - chromosomes = made of DNA and associated proteins (nucleoprotein) = chromatin - 2 types of proteins in chromatin: o 1) histones = most abundant in chromatin, small basic proteins 5 major types: H1 (H5 in RBC of birds), H2A, H2B, H3, H4 J distinguished by arginine/lysine ratio (high) - H3/H4 highly conserved in evolution (conventional histones) basic change allows interaction w/ negative charge of DNA J histones can be further modified by phosphorylation and acetylation (post-translational modification) histone variants thought to have specialized functions (e.g. H2A associated w/ inactivated X) o 2) non-histone proteins = diverse structural/enzymatic/regulatory protein, many yet to be characterized (include Tc factors) - when chromatin extracted from nuclei and examined by electron microscopy, appearance depends on salt concentration used to extract DNA o low salt (removal of H1) in absence of cations (Mg ) J beads on string, extended linker DNA connecting beadlike nucleosome (10nm diameter) 2+ o physiological salt (0.15M KCl, 0.004M Mg ) J condensed fiberlike form, 30nm diameter, not extended beads on string - when chromatin treated w/ nonspecific nucleases (DNAse 1), DNA converted to fragment J approx 200 bps - when treating naked DNA (no protein) J produce random sized fragments J so DNA in chromatin protected (by protein) from enzymatic attack except at periodic sites - nuclease digestion lead Kornberg to say DNA and histones organized into repeating subunits = nucleosomes - nucleosome = lowest level of chromosomal organization, 200 bp repeats but repeats can vary o ordered packaging depends on histones o consists of nucleosome core particle w/ 146 bp of supercoiled DNA wrapped 1.8 times around disc-shaped complex of 8 histones (octamer): 2 each of H2A, H2B, H3, H4 o linker DNA connects nucleosome J H1 is linker histone and binds to part of linker J linker more variable in size and can be digested in controlled nuclease digestion experiments (15-55 bp) - bp spaced 0.34nm so 200 bp would take approx 70 nm is fully extended J nucleosome occupied 10nm o therefore, packaging ratio of DNA of nucleosome in 7:1 Lecture #7: Higher Order DNA Structure J Chapter 12 - exact structure of 30nm fiber still unknown how linker DNA organized (Zigzag model and solenoid model) o linker increases packaging 6-fold relative to nucleosomes - solenoid model of 30nm condensed chromatin fiber J higher level of chromatin structure o histone tails needed for condensation J 30nm fiber increases packaging 6-fold relative to nucleosome and 40 fold in total o packaged in irregular solenoid structure w/ 6 nucleosomes/turn o H1 on inside of solenoid o chromatin in chromosomal regions not being actively transcribed mostly in condensed 30nm fiber form or higher order (looped domains) - DNA and core histones held together by non-covalentbonds w/ ionic between negatively charges phosphates of DNA and positively charged residues in histones J make contact where minor groove of DNA faces inward towards histone core J at about 10 bp intervals) - looped domains compacted into chromosomes during mitosis o 30nm compacted into series of supercoiled loops o attached at ends to proteins that are part of nuclear matrix J scaffold o 1 protein = Topo II for controlling degree of supercoiling/reducing tangling - after mitosis, mitotic chromosomes return to diffuse interphase condition = euchromatin - approx 10% of chromatin remains condensed throughout interphase = heterochromatin J reps inactivated/silenced DNA - constitutive heterochromatin = permanently silenced J e.g. around centromeres, telomeres, usually highly repeated simple sequence (satellite DNA) - facultative heterochromatin )]L ]]L ]LZZZ}}2[Zo]J e.g. one of X chromosomes in females transcriptionally inactive to ensure males and females have same number of active X chromosomes and synthesize same amount of product from X-linked genes in females o both X chromosomes become active in oogenesis (in germ cells before meiosis) J so females are mosaics where 1 cell can express maternal X and another cell can express paternal (different alleles) o inactivation happens in early embryogenesis J explainscolour blindness in males but not females and why multicolored (black and orange patches) calico cats are always female Gene Expression - bacteria gene expression highly regulated - rapid - gene expression regulation primarily at level of transcription - changes in gene expression needed for response to changes in nutritional/physical environments (e.g. change in oo[ZLK] lZ o }K}LLZ - genes that encode enzymes of metabolic pathways usually clustered together in functional complex = operon o in E. coli, half genes clustered in operons - transcription of operon and of isolated genes controlled by RNA polymerase and specific repressor and activator proteins - repressed gene expression = corresponding mRNA and encoded proteins synthesized at low rates/no synthesis - activated gene expression = both mRNAs and encoded proteins produced at higher rates - operons composed of: o promoter region = site where RNA Polymerase binds to DNA o operator region = beside/overlapping promoter J binding site for repressor protein (regulatory protein that
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