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Final

BIOB11H3 Study Guide - Final Guide: Leucine Zipper, Chemotherapy, Tubulin


Department
Biological Sciences
Course Code
BIOB11H3
Professor
Shelley A.Brunt
Study Guide
Final

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BGY10 t Final #1
Lecture #6: Nucleus t Chapter 12
- nucleus = site of storage for genetic material to be replicated/expressed
- nuclear envelope surrounds nucleus t double membrane barrier
- cell in interphase has:
o chromatin = chromosome as highly extended nucleoprotein fibers
o nuclear matrix = fibrillar network w/ protein t residual filamentous Protienaceous matrix
o nucleoli = 1+, electron-dense structure t 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 assembly of protein t 3000 pores/cell t 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 t mechanical support for envelopes and site of
attachment of chromatin
- nucleus has major control t 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 t 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 t stretches of positively charged AA t function as
Z[
- proteins that move out have nuclear export signals
- proteins (karyopherins) that function as transport receptors moving macromolecules across nuclear envelopes t
importins: move macromolecules in, exportins: move macromolecules out
- importing proteins from cytoplasm into nucleus t GTP bring protein (high in nucleus, low in cytoplasm) t
conformational change of transporter (open channel)
o Z}]uW]u}]vrltt binds to protein/cargo to be transported
o accessory RCC1 in nucleus converts Ran-GDP to Ran-GTP, maintaining gradient
o ]u}]vtu}Àl]v}Ç}ouZ}Zv-GTP
o release w/ hydrolysis t needs accessory Ran-GAP1
- RNA usually move thru NPC as ribonucleoprotein complex (RNP) (except tRNA t recognized directly by exportin-t)
o RNP carries nuclear export signal t recognized by exportins
o mRNAs move 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) t 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 t expands volume
- metaphase chromosome = X shaped kind
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o centromere = has AT rich DNA (satellite) randomly repeated (constitutive heterochromatin) t 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 RNAs and 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 t distinguished by arginine/lysine ratio
(high) - H3/H4 highly conserved in evolution (conventional histones)
basic change allows interaction w/ negative charge of DNA t 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 (Mg2+) t beads on string, extended linker DNA connecting
beadlike nucleosome (10nm diameter)
o physiological salt (0.15M KCl, 0.004M Mg2+) t condensed fiberlike form, 30nm diameter, not extended beads
on string
- when chromatin treated w/ nonspecific nucleases (DNAse 1), DNA converted to fragment t approx 200 bps
- when treating naked DNA (no protein) t produce random sized fragments t 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 t H1 is linker histone and binds to part of linker t 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 t nucleosome occupied 10nm
o therefore, packaging ratio of DNA of nucleosome in 7:1
Lecture #7: Higher Order DNA Structure t 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 t higher level of chromatin structure
o histone tails needed for condensation t 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-covalent bonds w/ ionic between negatively charges phosphates of
DNA and positively charged residues in histones t make contact where minor groove of DNA faces inward towards
histone core t 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 t 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 t reps inactivated/silenced
DNA
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- constitutive heterochromatin = permanently silenced t e.g. around centromeres, telomeres, usually highly
repeated simple sequence (satellite DNA)
- facultative heterochromatin A]v]À]v]vZ}(}P[o](t 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) t 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 t explains colour 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[vÌÇu]lµµo}u}vv
- 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 t binding site for repressor protein (regulatory protein that
recognizes specific nucleotide sequence)
o regulatory gene = encodes regulatory protein that usually has own promoter and not contiguous/close w/
structural genes
o structural genes = encode enzymes t transcribed into single mRNA (polycistronic) and translated into
individual enzymes of metabolic pathway
- inducible operon t e.g. lactose (lac) operon:
o controls expression of genes for utilization of disaccharide, lactose t t-galactosidase ~t-gal) = enzyme
responsible for hydrolysis of bond that joins 2 sugars in lactose (glucose and galactose)
o ]vv}(o}U(Á}}(vÌÇuUt-gal produced
o more lactose causes more enzyme production = inducible operon
o under positive control
o CAP = catabolite activator protein t or cAMP receptor binding protein
o glucose effect (sugar used 1st), low cAMP concentration in presence of glucose
o cAMP-CAP (CRP) complex binds when cAMP high t enhances Tc b/c changes DNA conformation
- repressible operon t e.g. tryptophan operon:
o produces enzymes needed for manufacture of tryptophan
o no tryptophan: bacteria expends energy to make AA (tryptophan)
o presence of tryptophan causes shut down in enzyme expression for tryptophan = repressible operon
binding of repressor to tryptophan (co-repressor) needed to inactivate operon
v}(Ç}ZvÇvZ]}(vÌÇu]vÀ}oÀ]v][}ÁvÇvZ]
conformation changes to repressor critical for Tc activation
- for both operons (lactose/tryptophan), promoter/operator works in cis and repressor works in trans
- proteins synthesized by bacterial cell usually determined by conditions of cell growth t cell/tissue specific expression
not involved
o bacteria express approx 3000 genes in totally and 1000 genes at any time
- more eukaryotes composed of different cell types = multi-cellular and have specialized activities needing differential
expression:
o during development
o cell specific gene expression
o responses to environmental stimuli
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