Unit II- Nuclear Structure and Function

Question 1

What are the functions of the nucleus?

Answer 1

1) Protect the chromosomes from damage
2) Repair the chromosomes when damaged
3) Replicate the chromosomes
4) Facilitate the expression of genetic programs through the transcription of gene sets
5) Synthesize ribosomes

Question 2

Overview of nuclear structure

Answer 2

• nucleus surrounded by a cage of intermediate filaments
• enclosed by a doubled membrane called nuclear envelop
• nuclear envelop punctuated by nuclear pores that are conduits through which all traffic into and out of the nucleus occurs
• chromosomes of interphase cells are collectively visualized as two forms of chromatin
  
  • euchromatin stains more lightly and is less condensed DNA that is being expressed
  • heterochromatin stains more darkly and is more condensed DNA this is not in the process of being expressed
• the nucleolus is a non-membrane bounded sub-compartment of the nucleus where ribosome biogenesis occurs

Question 3

Nuclear envelope

Answer 3

• double membrane structure that forms a barrier between the nucleus and cytoplasm
• outer membrane is continuous with the membrane of the ER and has ribosomes attached to it
• proteins translated on the outer membrane are inserted into the lumen between the outer and inner membranes
• the inner membrane is attached to and supported by nuclear lamina

Question 4

Nuclear lamina

Answer 4

• a meshwork of intermediate filaments
• made by oligomerization of the intermediate filament proteins lamin A, B, and C
• it is attached to the inner membrane of the nuclear envelop and helps to stabilize the nuclear envelop and organize chromosomes
• phosphoylation of the lamin proteins at mitosis induces disassembly of the nuclear lamina which in turn causes nuclear breakdown

Question 5

Nuclear Dynamic During Mitosis

Answer 5

Before:

• transcription ceases
• nucleolus dissolves
• chromosomes condense
• modification of the lamins causes the nuclear envelope to break down into vesicles

After:

• Lamin A is dephosphorylated
• nuclear envelope vesicles fuse onto decondensing chromosomes
• chromosomes fuse
• transcription of rDNA repearts to re-formation of the nucleolus first as 10 pre-nucleolar foci

Question 6

Nuclear Pores

Answer 6

• proteinaceous channels through the nuclear envelope
• conduits through which all molecules move into and out of the nucleus
• form a 9nm in diameter, semi-selective channel; solutes and small proteins can diffuse through, the transport of anything larger is facilitated
• ~125 Mega Daltons ~100 proteins arranged in 8-fold symmetry
• most cells have 3-4,000, functionally equivalent NPCs

Question 7

General Mechanism of Transport for both Export and Import

Answer 7

-the process is facilitated by carrier proteins that recognize transport substrates via signals on the substrates: nuclear localization signals for import and nuclear export signals for export. The carrier binds the substrate and acts as an escort through the pore

Question 8

The Role of Ran in Transport

Answer 8

• the energy for transport comes from GTP hydrolysis by the G-protein Ran. Ran-GDP predominates on the cytosolic face while Ran-GTP predominates on the nuclear face.
• due to asymmetric localization of the Ran GAP and Ran GEF respectively

Question 9

Nuclear Localization SIgnals

Answer 9

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

Question 10

Protein import

Answer 10

• NLS on protein is recognized and bound by importin
• importin directs docking of the complex onto fibrils extending from the cytoplasmic face of the NPC (Nuclear Pore Complex) followed by transfer to NPC proper
• the cargo-carrier complex is translocated through the NPC
• GTP-bound Ran directs dissociation of the carrier from the cargoand release of the NLS-containing proteins into the nucleus
• the importin bounded by Ran GTP and then is transported back across to cytosol and there is GTP hydrolosis to make Ran GDP

Question 11

RNA export

Answer 11

• mRNA, tRNA, and rRNA exported from the nucleus in a very similar mechanism to protein import
• the substrate for export is recognized by a carrier protein called exportin and Ran-GTP (in mRNA the exportin recognized the 5’ CAP)
• In cytoplasm GTP hydrolysis causes the cargo to dissociate and exportin is transported back into nucleus

Question 12

The Role of Ran GTPase

Answer 12

• provide the energy for transport in the form of potential energy 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 13

The Nucleolus

Answer 13

• 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

1) Fibrillar Center- this region contains DNA that is not being actively transcribed
2) Dense fibrillar component-this region contains rRNA molecules actively being synthesized
3) Granular Component- this region contains maturing ribosomal precursor particles

Question 14

Transcription of rDNA genes

Answer 14

• 3 of 4 rRNA species (28S, 18S, and 5.8S) are transcribed as a large 45 S precursor
• ~200 rDNA genes per haploid genome are located in tandem arrays on 5 different chromosomes, 2 homologues each =10
• ~100 transcription units/gene are simultaneously transcribed by RNA Pol I

Question 15

45 rRNA precursor processing

Answer 15

• 13,000 nucleotide precursor is processed in the pre-ribosome by endo- and exonucleases to 28S, 18S, 5.8S rRNAs
• 18S is incorporated into the small subunits, the 28S, 5.8S, and 5S (made elsewhere by RNA polymerase III) are incorporated into the large ribosomal subunit

Question 16

Ribosome biogenesis pathway

Answer 16

• ~80 ribosomal proteins assemble on the 45S precursor
• 45S rRNA is processed in the particle to create the immature small and large sub-units
• process is completed following export from the nucleus

Question 17

Human Chromosomes

Answer 17

• extremely long (1.7-8.5 cm), linear polymers of double stranded DNA
• 22 different autosomes and 2 different sex chromosomes
• diploid cells have 44 autosomes and 2 sex chromosomes; XX= female, XY= male
• haploid genome contains ~3x10^9 base pairs (~1,000 times the size of E.coli genome)
• chromosomes are coated with both structural and regulatory proteins = cromatin
• the human genome has been sequenced

Question 18

Elements required for stability and propagation of chromosomes

Answer 18

• 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 19

Replication of Telomeres

Answer 19

• the ends of lagging strands cannot be primed
• a specialize enzyme called telomerase catalyzes the synthesis of several repeats of the sequence GGGGTTA
• An RNA in telomerase provides the template for the repeat

Question 20

Information on chromosomes

Answer 20

-surprisingly little of the sequence of the human genome codes for anything.
- genes and junk DNA
Junk DNA:
-alu sequences-old non-functional transposons, highly repetitive and ~5% of the genome
-transposons-mobile DNA elements, ~34% of the genome
-pseudogenes-non-functional gene replicates

Question 21

“Split” Genes

Answer 21

• gene code for RNAs of which the major classes are rRNA, tRNA, and mRNA
• other classes of RNAs include snRNAs and snoRNAs
• more recently a new class of regulatory RNAs, called micro-RNAs have been discovered
• in mRNA, most of the precursor RNA is removed by a process called splicing that joins exons and removed introns
• this exon/intron system has played a critical role in the evolution of genes

Question 22

Chromosome Packaging

Answer 22

Two levels of packaging in interphase cells:

• euchromatin = less condensed, actively transcribed DNA
• heterchromatin= more condensed and not being expressed

Packaging is mediated by the histones:

• histones are abundant, positively charged DNA binding proteins
• there are 5 kinds of major histones: H1, H2A (H2AZ), H2B, H3, and H4 involved in elementary chromosome packaging

-at mitosis- chromosome condensation

Question 23

The Nucleosome

Answer 23

• a histone octomer is formed from 2 copies each of H2A, H2B, H3 and H4
• 146 bp’s of DNA is wrapped around the barrel-shaped octamer
• including linker DNA 200 bps of DNA is packaged per nucleosome, leading to a reduction in length to ~1/3 of linear

Question 24

The 30-nm chromatin fiber

Answer 24

• one copy per nucleosome of histone H1 binds the linker DNA and stabilizes the nucleosome into a 30 nm fiber
• the 30 nm fiber is disrupted by regulatory proteins to allow for transcription of genes

Question 25

Chromosome looping

Answer 25

- additional levels of packaging are poorly understood - involves organization of the chromosomes into loops - at mitosis the chromosomes condense, achieving their highest level of packaging

Question 26

Epigenetics and the histone tails

Answer 26

- -the histone N-terminal tails protrude from the 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 accesibility to, or the recruiting of, regulatory proteins

Question 27

Nuclear Architecture

Answer 27

- the nuclear matrix is an underlying fibrous network that facilitates further organization of the nucleus - chromosomes are attached to the nuclear matrix cia Matrix- Associated Regions (MARs_ - euchromatin is organized into a small number of foci - there are opens channels to the nuclear pores

Question 28

The Nucleus and Disease | Lupus

Answer 28

Lupus: Auto-immune disease were patients produce auto-reactive antibodies to nuclear antigens

Question 29

Acute Promyelocytic Leukemia

Answer 29

-severe bleeding disorder due to a reciprocal translocation that fuses the retinoic acid receptor to the nuclear protein PML creating altered activities

Question 30

Spinal Muscular Atrophy

Answer 30

childhood neuromuscular disease | -inherited mutation in SMN1 an RNA processing factor