Unit 6: The Nucleus (a) Flashcards
(45 cards)
1
Q
*What are the functions of the nucleus?
A
- Serve as storehouse for *storehouse for genetic information
- At genomic level:
- *DNA replication
- *RNA transcription + processing
- *Regulates gene expression (by regulating transport of tf from cytoplasm to nucleus)
2
Q
*What is the role nuclear envelope?
A
- Acts as a *selective barrier: prevents free traffic between molecules in nucleus + cytoplasm. *Nuclear pore complexes constitute only communication channels between the nucleus + cytoplasm, allowing the controlled exchange of molecules between both compartments.
- maintains both compartments *metabolically independent
- Maintains internal composition of nucleus
- Key role in regulation of gene expression in eukaryotes
- Controlled transcription by regulating transport of tf to nucleus
- Post transcriptional mechanisms (e.g. alternative splicing)
3
Q
*What does the nuclear envelope structure consist of?
A
- *2 Nuclear membranes (outer+inner)
- *Nuclear pore complex
- *Nuclear lamina (underlies the INM)
*See pg. 6 for electron micrographs and learn how to label
4
Q
*What is the role of the outer nuclear membrane?
A
- Continuation with membrane of endoplasmic reticulum
- Communication between nuclear inter membrane space + lumen of endoplasmic reticulum
- Functionally similar to endoplasmic reticulum
- Has ribosomes on surface + membrane proteins that bind to cytoskeleton
5
Q
*What is the role of the inner nuclear membrane?
A
- Has integral membrane proteins specific to nucleus (some bind to lamina)
6
Q
*What is the role of the nuclear pore complex?
A
- Inner + outer membrane join together in nuclear pore complex
- The only channels that allow small polar molecules + macromolecules pass through the nuclear envelope
- Complex structure responsible for selective trafficking of proteins + RNA between nucleus + cytoplasm
*See pg. 8 for electron micrograph + diagram on pg. 14
7
Q
*What is the role of the nuclear lamina?
A
- Fiber network providing structural support to nucleus
- Made up of lamin A, B + C proteins
- All lamina are fibrous proteins between 60-80 kDa
- Lamins joined together to form filaments
- see pg 9 for micrograph
kDa=kilo Daltons (dalton= atomic mass unit for proteins, 1aa= 110 Da)
8
Q
*What is the 1st level of association?
A
- The interaction between 2 lamins to form a *dimer.
- α-helix areas of 2 polypeptides wrap around each other forming a coiled coil structure
9
Q
*How do dimers associate with each other?
A
- Head to tail forming *filaments
10
Q
*How is the nuclear lamina formed?
A
- Filaments formed from dimers interact with each other to form the nuclear *lamina
11
Q
*What assists interactions between the lamina + inner nuclear membrane?
A
- *Post-translational addition of lipids
- *Interaction with integral proteins (from inner membrane) e.g. emerin, LBR (lamin B receptor) or SUN proteins
12
Q
How does the addition of post-translational lipids + integral proteins (from inner membrane) facilitate interaction between lamina + inner membrane?
A
- SUN proteins bind to KASH proteins of outer nuclear envelope, creating LINC complex connecting the lamina with cytoskeleton
- Lamina + inner membrane proteins interact via emerin + LBR with chromatin associated proteins
13
Q
What is the structure of the central channel?
A
- 8 protein spokes around central channel
- spokes attached to cytoplasmic ring + another nuclear ring
- spoke-ring structure anchored to nuclear envelope at fusion sites between inner+outer membranes
- cytoplasmic filaments exend from nuclear ring, forming nuclear basket
- during passage of macromolecules, pore channel can be modified from 9nm to 40nm
14
Q
How is traffic between the nucleus + cytoplasm controlled?
6.2
A
- 2 mechanisms *depending on size of molecule
- *Passive diffusion: small molecules+ proteins with atomic weight less than 40kDa pass through both directions
- *Selective transport: Most RNA + proteins cross nuclear pore complex using this mechanism, molecules are recognised and selectively transported in specific direction
15
Q
How does selective transport work?
A
- Proteins needed for nuclear functions enter nucleus from synthesis sites in the cytoplasm
- Many proteins undergo continuous transfer
- Proteins responsible for structure + function of genome ‘tagged’ to be destined for nucleus with specific aa sequences called *NLS (nuclear localisation signals), which are recognised by nuclear transport receptors
Histones, DNA/RNA polymerases, tf etc. through this method
16
Q
*Who first characterised the NLS?
A
*Alan Smith et al. (1984) by studying the T antigen of the simian virus SV40
17
Q
How did Alan Smith’s experiment show NLS?
A
- T antigen is a virus encoded protein that initiates viral DNA replication in infected cells
- Protein is located in nucleus
- 1st experiment shows Lys-128 mutation prevented T antigen accumulation in the nucleus, accumulating in the cytoplasm instead => suggests Lys-128 as part of NLS
- Later shown that a 7aa sequence (residue 126 to 132) was responsible for NLS of T antigen
18
Q
- What did targeted deletion experiments show?
6. 2
A
- Through elimination of aa in the protein, they found deletions compromising residues 1-125 or between 133 + carboxyl terminal end of T antigen, The accumulation was normally in the nucleus
- Deletions from aa 126-132 caused retention of T antigen in cytoplasm
- Found sequence of residues 126-132 added, through creation of chimeras, to cytoplasmic proteins (beta-galactosidase + pyruvate kinase), caused accumulation in nucleus (pic of pg.20)
19
Q
What other proteins has NLS been identified in?
A
- NLS of SV40 T antigen found to be a prototype for similar sequences of other nucleus proteins
- Most sequences are short, rich in Lys + Arg + aa. Responsible for nuclear signaling are contiguous with each other
- In other cases the aa are together, but not necessarily contiguous. As is the case with *nucleoplasmin (protein that participates in the assembly of chromatin) in with the NLS is a *bipartite sequence, formed by a Lys-Arg sequence separated by *10 aa of the sequence *Lys-Lys-Lys-Lys
20
Q
*What are the 2 proteins that have a role in the transport of proteins to the nucleus?
A
- *Importins: recognise NLS of the protein-cargo and transport it from the cytoplasm to the nucleus
- *Ran proteins:
- Guanosine di/triphosphate (GDP/GTP) binding protein
- Conformation + activity regulated by being bound to GTP or GDP
- High conc. of Ran/GTP in nucleus, determines directionality of nuclear transport
- Enzymes that stimulate exchange of GDP for GTP on Ran are located on nuclear side of nuclear envelope; while those that stimulate GTP hydrolysis found on cytoplasmic side.
21
Q
How do the 2 proteins play a role in the transport of proteins to the nucleus ?
A
- Importin recognises NLS of cargo protein.
- Cargo-importin complex binds to proteins of cytoplasmic filaments of nuclear pore complex and is transported through pore.
- In nucleus, Ran/GTP binds to importin, disrupting the cargo-importin complex + releasing cargo protein.
- Importin-Ran/ GTP complex is re-exported through nuclear pore.
- The GTPase activating protein (Ran-GAP) associated with cytoplasmic filaments hydrolyses GTP in Ran to GDP, releasing importin.
- Ran/GDP is transported back to nucleus associated with its own import receptor: NTF@
- In nucleus, Ran-GEF (chromatin bound) causes Ran-bound to GDP to be exchanged for GTP, regenerating Ran/GTP
Video on pg.25
22
Q
*How are proteins exported to the cytoplasm?
A
Proteins are tagged with specific aa sequence called *NES (nuclear export signal), often rich in leucine
23
Q
How does the NES work?
A
- signals recognised by *exportins (receptors within nucleus) that direct transport of proteins to the cytoplasm through nuclear pore complex
- Cargo-exportin complex binds to Ran/GTP, which directs the movement of proteins with NES from nucleus to cytoplasm
- Once transport to cytosolic side occurs, hydrolysis of GTP causes dissociation of target protein, which is released into cytosol.
- Exportins + Ran/GDP recycled through nuclear pore complex
24
Q
*What mechanisms regulate transport to the nucleus?
A
- Some *cytoplasmic proteins mask NLS, causing the proteins to remain in cytoplasm
e. g Tf *NF-kB in un stimulated cells is bound to an inhibitor protein (IkB) that masks the NLS. In stimulated cells, IkB is phosphorylated + degraded, allowing transport of NF-kB to nucleus - Other proteins are restricted from entering the nucleus by *phosphorylation
e. g. Tf Pho4 is phosphorylated on a serine residueadjacent to the NLS, preventing its binding to importin. Regulated dephosphorylation exposes NLS + allows Pho4 to be transported to nucleus at appropriate stages of cell cycle
25
*How does RNA transport occur?
- RNA involved in protein synthesis exported from nucleus
- RNA transported through nuclear envelope as *ribonucleoprotein complexes (RNPs)
- Export of *tRNA, *miRNA + *rRNA: mediated by specific exportins + Ran/GTP
- tRNA + miRNA precursors: *exportin-t + *exportin-5 respectively, bind to RNA directly
- rRNAs first associate with ribosomal proteins in nucleolus, nascent 40S + 60S ribosomal subunits transported separately to cytoplasm by exportin *Crm1
- *mRNA export. Exportins + Ran not involved. After mRNA processing, an *exporter complex made of various preoteins transports mRNA. A helicase associated with cytoplasmic face of CNP releases mRNA into cytoplasm
26
*What do other small non coding RNAs do in the nucleus?
- *snRNAs involved in splicing of pre-mRNAs
- *snRNAs initially transported to cytoplasm by *Crm1, where they bind to proteins to form functional RNPs that are carried to nucleus by importin *snuportin
- snoRNAs are involved in rRNA processing
- *snoRNAs remain in nucleus
27
* How is chromatin organised during interphase?
| 6. 3
- Most chromatin decondeses + distributed throughout nucleus, *not randomly
- each chromosome occupies region of nucleus
- organised so transcriptional activity of gene is related to position
- main activities of DNA replication + transcription take place in clustered regions within nucleus
28
What are chromosome territories?
- Non-random distribution of interphase chromosomes suggested in *1886 by *Carl Rabl
- Proposed each chromosome occupies specific area with centromeres + telomeres attached to opposite sides of nuclear envelope
- Model confirmed in 1984 in studies in Drosophilia. Each chromosome occupies specific place inside nucleus: *chromosome territories
29
Where are genes located in the nucleus of mammalian cells?
- Also occupy different areas of nucleus
- Genes transcribes believed to be located on peripheries of these areas, close to channels that separate chromosomes (*interchromosomal domains), where RNA processing + transport occur
- Recent analysis show RNA transcripts are generated within territories + are traversed by channels communicated with the non-chromatin regions
*See diagram on pg.34 on “Organisation of chromosomes in the nuclei of mammals”
30
*How does transcriptional activity affect location of chromatin?
- *Euchromatin: most chromatin of interphase cells. Decondensed + transcriptionally active. Preferably located inside nucleus
- *Heterochromatin: highly condensed that is not transcribed. Often associated with nuclear envelope or periphery of nucleolus
- * Constitutive Heterochromatin: DNA segments that are always and in all cell types in condensed form e.g. highly repetitive sequences in centromeres + telomeres
- * Facultative Heterochromatin: Genes that are not transcribed in that cell type or at that time in cell cycle
- Chromosomes rich in genes located in center while those with few in the peripheries
31
What is the Barr corpuscle?
- transcriptional inactivation
- in cells of female mammals
- Heterochromatin region next to nuclear membrane
- corresponds to portion of 1 of X chromosomes, inactivated by high degree of packaging
- Goal: compensate for genetic dose (X chromosome= >100 genes; Y chromosome= <100)
- inactivation occur in embryonic development + is random
- Heterochromatisation reverts in germ cells, so haploid oocytes contain X chromosome
Oocyte= immature egg
32
What are replication factories?
- Nucleus regions where there are complexes of *proteins involved in *replication or transcription
- *Replication factories: discrete regions where DNA replicates
- each site contains 5-50 replication forks
- newly synthesised DNA
- proteins involved in replication
e. g. PCNA-GFP. Proliferating cell nuclear antigen (PCNA) is a nuclear protein that promotes DNA synthesis, as it is cofactor of δ-DNA polymerase
33
What are transcription factories?
- Transcription factories: discrete regions where DNA is transcribed to RNA
- Contain newly synthesised RNA and highly enriched with active RNA polymerases + tf
- Several chromosomal loci can share same transcriptional factory
- Co-regulated genes can be transcribed in the same factories, thus transcription factories facilitate coordinated regulation of related genes
e. g. immunoglobulin genes from different chromosomes located in same transcription factory in lymphocytes that produce large amounts of antibodies
34
What are nuclear bodies?
| 6.4
- differentiated organelles in nucleus
- Compartmentalise nucleus and serve to concentrate RNA + proteins that function in certain processes in the nucleus
- Do not have membranes, maintained by interactions between proteins and between proteins + RNA.
- Dynamic structures capable of exchanging their content with rest of nucleus
- Active field of research: function not fully known
35
*What are Cajal bodies?
- Described by Ramon y Cajal in 1906
- intervene in *final stages of maturation of snRNP (small nuclear ribonucleoproteins, formed by snRNA associated with proteins):
modification of snRNAs by ribose *methylation
*pseudouridylation: conversion (isomerisation) of uridine to a diff. isomeric form, pseudouridine
36
What are speckles?
- *Components of the splicing system are conc. in 20-50 discrete structures, speckles
- After assembly + maturation in Cajal bodies, snRNP transferred to nuclear speckles, where splicing factors are found
- Genes actively transcribed are distributed throughout nucleus, but components of splicing machinery are conc. in these discrete domains
- Structures are storage site for components responsible for splicing, from her recruited to actively transcribed genes, where pre-mRNA processing occurs.
Localisation of these components in discrete domains has been carried out by immunofluorescence using antibodies against *ribonucleoproteins (RNPsn) + *splicing factors
37
What is the nucleolus?
- largest structure in nucleus (eukaryotic)
- Site of ribosome biogenesis. *Transcription + processing of *28S, *5.8S + 18S rRNA takes place + *assembly of ribosomes
- 3 rRNAs transcribes as single unit in nucleolus by RNApoll, gibing rise to 45S pre-rRNA, processed into these 3 rRNAs
- 5S rRNA transcribed outside nucleolus by RNApollll
38
*How many ribosomes do continuously growing mammalian cells contain?
- *5 million-10 million which must be synthesised each time the cell divides.
- Cells contain *multiple copies of rRNA genes to meet transcription demand of large number of rRNA molecules
39
*What are the nucleolus organisation regions?
The nucleolus is organised around the chromosomes that contain genes for the 5.8S, 18S + 28S, called nuclear organisation regions
40
*How does transcription of rRNA genes take place?
- 3 rRNA genes separated by spacer DNA that is not transcribed
- High density of growing RNA chains is due to large number of RNA polymerase molecules, present at maximum density, approx. 1 polymerase per 100 bp of template DNA
41
What are the 3 distance regions of the nucleolus?
- *The fibrilar center (FC: where genes that encode rRNA are located, transcribes at the interface with DFC
- *The dense fibrilar component (DFC): where the pre-rRNA is processed
- *The granular component (G): where ribosomal subunits are assembled
- Size of nucleolus depends on metabolic activity of cell. Large present in cells actively engaged in protein synthesis. Variation due to differences in granular component size, which reflects rate of ribsosome assembly
See pg. 37 for micrograph
42
*How is rRNA transcribed + processed?
- The large 45S pre-rRNA is processed through series of cleavages
- *Outer (ETS) that are transcribes are located at 5’ and 3’ end of pre-rRNAs, and 2 *internal spacers (ITS) between 18S, 5.8S + 28S rRNA sequences
- 1st cleavage occurs within ETS near 5’ end of pre-rRNA, during early stages of transcription
- Once transcription is complete, ETS is eliminated at 3’
- Subsequent cleavages give rise to mature rRNAs
43
*How is pre-rRNA processing involved in important nucleotide modifications?
- Addition of *methyl groups to ribose residues
| - Conversion of uridine to *pseudouridine
44
Why does pre-rRNA processing require intervention of proteins + RNA in nucleolus?
- Nucleolus contains more than 300 proteins + approx. 200 snoRNA (small nucleolar RNAs) that are involved in pre-rRNA processing
- snoRNAs with the proteins form snoRNPs that bind to pre-rRNA to form a processing complex.
- snoRNAs contain short sequences (15nt approx.) complementary to rRNA and by base pairing, direct enzymes that catalyse rRNA modification (e.g. methylation) to appropriate places
45
How does ribosome assembly occur?
- Involves assembly of pre-rRNA with ribosomal proteins + 5S rRNA
- Genes encoding ribosomal proteins transcribed outside nucleolus by *RNA polymerase II + translated in cytoplasm
- Ribosomal proteins transported to nucleolus where they assemble with pre-rRNA to form *preribosomal particles
- Association of ribosomal proteins with rRNA begins while synthesis of rRNA occurs. More than half ribosomal proteins are bound to pre-rRNA prior to processing.
- Remaining ribosomal proteins + 5S RNA are incorporated into preribosomal particles while cleavage of rRNA takes place.
