Nuclear Structure and Function

Question 1

Functions of the nucleus

Answer 1

• protect chromosomes from damage
• repair chromosomes when damaged
• replicate chromosomes
• facilitate the expression of genetic programs through the transcription of gene sets
• synthesize ribosomes

Question 2

nuclear structure

Answer 2

• surrounded by cage of IF and enclosed by double membrane nuclear envelope
• envelope punctuated by nuclear pores that are the conduits for traffic of everything in and out

Question 3

chromatin

Answer 3

complex of protein and DNA

Question 4

euchromatin

Answer 4

-stains more lightly and is less condensed DNA that is being actively expressed

Question 5

heterochromatin

Answer 5

-stains more darkly and is more condensed DNA that is not in the process of being expressed

Question 6

nucleolus 1

Answer 6

non membrane bound subcompartment of the nucleus where ribosome biogenesis occurs

Question 7

nuclear envelope

Answer 7

• double membrane
• outer membrane continuous with membrane of ER and has active ribosomes attached to it
• proteins translated on outer membrane are inserted into the lumen b/n membranes
• inner membrane attached to and supported by nuclear lamina

Question 8

nuclear lamina

Answer 8

• meshwork of IF
• lamin A, B, C
• attached to inner membrane of nuclear envelope and helps stabilize nuclear envelope and organize chromosomes
• phosphorylation of lamins at mitosis induces disassembly of the nuclear lamina which causes nuclear breakdown

Question 9

nuclear dynamics during mitosis

Answer 9

before:
-transcription ceases
-nucleolus dissolves
-chromosomes condense
-modification of lamins
after:
-lamin A dephosphorylated
-nuclear envelope vesicles fuse onto decondensing chromosomes
-chromosomes fuse
-transcription of rDNA repeats leads to re-formation of nucleolus

Question 10

nuclear pores (NPCs)

Answer 10

• proteinaceous channels through the nuclear envelope
• conduits through which all molecules move into and out of the nucleus
• form a 9mm diameter, 15nm length, semi-selective channel
• solutes and proteins (<44kDa can diffuse alone) is facilitated
• ~125 mega daltons, ~100 proteins arranged in 8-fold symmetry
• most cells have 3-4,000 functionally equivalent NPCs

Question 11

general mechanism of transport in and out of nucleus

Answer 11

• process is facilitated by carrier proteins that recognize transport substrates via signals on substrates.
• nuclear localization=import
• export signals
• carrier binds substrate binds and acts as escort through the pore

Question 12

Ran in transport

Answer 12

• energy comes from GTP hydrolysis by Ran
• Ran-GDP on cytoplasmic side (GAP changes GTP to GDP when it gets to cytosolic side)
• Ran-GTP on nuclear side (GEF changes GDP to GTP when it gets to nuclear side)
• creates PE gradient

Question 13

nuclear localization signal

Answer 13

• required to recognize and direct proteins for import into the nucleus. recognized by importin complex
• 4-8 positively charged amino acids (lysine and/or arginine)
• can occur anywhere in primary sequence

Question 14

detailed mechanism of transport-import

Answer 14

1. cargo binds to importin
2. importin-cargo complex diffuses through pore
3. Ran-GTP binds to complex, causing a release of cargo
4. Ran-GTP and importin diffuse back across pore, and GAP helps hydrolysis to Ran-GDP and release of importin
   * Ran GDP cycled back into nucleus and GEF changes it to GTP for next cargo-importin complex

Question 15

detailed mechanism of transport-export

Answer 15

1. cargo and Ran GTP bind to exportin
2. diffuse through NPC
3. Ran-GTP hydrolysis by GAP releases cargo and exportin.
4. Exporting and Ran-GDP cycled back across

Question 16

GAP and GEF

Answer 16

GAP in cytoplasm
GEF in nucleus

Ran-GDP moves from cytoplasm to nucleus, is GEFed to Ran-GTP, moves back, and is hydrolyzed with help of GAP

Question 17

Ran GTPase

Answer 17

• provides energy for transport in the form of PE stored as a chemical gradient
• it helps form export complexes and accompanies these complexes out of the nucleus
• catalyze release of the transport complex after transport and thus ensures irreversibility

Question 18

nucleolus 2

Answer 18

• non membrane bound sub-compartment of the nucleus
• assembled on the rDNA repeats of 5 different autosomes with two homologues each for a total of 10 chromosomes
• primary function is ribosome biogenesis

Question 19

three regions of the nucleolus

Answer 19

• fibrillar center-contains DNA that isn’t being actively transcribed
• dense fibrillar component-contains rRNA molecules being actively synthesized
• granular component-maturing ribosomal precursor particles

Question 20

transcription of rDNA genes

Answer 20

• 3 of 4 rRNA species are transcribed as a large 45S pre-cursor
• ~200 rDNA genes per haploid genome are located in tandem array on 5 chromosomes (2 homologues= 10)
• ~100 transcription units/gene are simultaneously transcribed by RNA Pol I.

Question 21

45S rRNA precursor processing

Answer 21

• 13,000 nucleotide precursor is processed in the pre-ribosome by endo and exonucleases to 28S, 18S, and 5.8S rRNAs
• 18S is incorporated into the small subunit
• 28S, 5.8S, and 5S (from RNA Pol III somewhere else) are incorporated into the large subunit

Question 22

Ribosome biogenesis

Answer 22

• about 80 ribosomal proteins assemble on the 45S precursor (and the 5S)
• 45S rRNA is processed in the particle with nucleases to create the other sized S rRNAS and immature small and large sub-units
• process is completed following export from the nucleus

Question 23

human chromosomes

Answer 23

• extremely long (1.7-8.5cm), linear polymers of double stranded DNA
• 22 different autosomes and 2 different sex chromosomes
• diploid cells have 44 autosomes and 2 sex chromosomes
• haploid genome contains ~3x10^9 base pairs (1000x size of E. coli)
• chromosomes coated in structural and regulatory proteins- chromatin
• human genome has been sequenced

Question 24

elements required for stability and propagation of chromosomes

Answer 24

• replication origins-initiation of DNA replication (~100,000)
• centromeres-segregation of duplicated chromosomes at mitosis and meiosis
• telomeres-stability and replication of the chromosome ends

Question 25

information content on chromosomes

Answer 25

- surprisingly little of the sequence codes for anything. that which does code is broken down into functional unit called gene - other substance includes: Alu sequences, transposons, and pseudogenes

Question 26

split genes

Answer 26

- genes code for RNAs- rRNA, tRNA, mRNA - other classes include snRNA and snoRNA - recently discovered micro-RNA - in mRNA, most of the precursor is removed by splicing that joins exons and removes introns - exon/intron system plays critical role in evolution of genes

Question 27

chromosome packaging

Answer 27

- mediated by histones (pos charge)- highly conserved - abundant, positively charged DNA binding proteins - 5 kinds- H1, H2A (H2AZ) H2B, H3, H4 - mitosis has high level of packaging called chromosome condensation

Question 28

nucleosome

Answer 28

- histone octamer - formed from 2 copies each of h@a, H2B, H3. H4 - 146 bp's of DNA is wrapped around the barrel-shaped octomer - including linker DNA, 200 bps of DNA is packaged per nucleosome, leading to a 1/3 linear reduction of length

Question 29

30 nm chromatin fiber

Answer 29

- one H1 per nucleosome coils and binds the linker DNA and stabilizes nucleosomes in 30 nm fiber - disrupted by regulatory proteins to allow for transcription of genes

Question 30

chromosome looping

Answer 30

- additional levels of packaging not well understood - involves organization of chromosomes into loops - at mitosis the chromosomes condense, achieving highest level of packaging

Question 31

epigenetics and histone tails

Answer 31

- histone N terminal tails protrude from nucleosome - lysine residues in the tails can be modified by methylation and acetylation - patterns of acetylation are regulated and are metastable in that they can persist through many cell divisions - acetylation locally alters chromatin structure and protein composition affecting accessibility to, or the recruiting of regulatory proteins

Question 32

chromosome organization

Answer 32

-fractal globules

Question 33

nuclear architecture

Answer 33

- nuclear matrix is an underlying fibrous network that facilitates further organization - chromosomes are attached to the nuclear matrix via Matrix associated Regions (MARs) - euchromatin is organized into a small number of foci - there are open channels to the nuclearpores

Question 34

nucleus and diease

Answer 34

- lupus-AI disease produce antibodies to nuclear antigens - acute promyelocytic leukemia-reciprocal translocation that fuses the retinoic acid receptor to the protein PML creating altered activites - spinal muscular atrophy-childhood neuromuscular disease. inherited mutation in SMN1-RNA processing factor