Genome organisation, rRNA processing and Nuclear Transport Flashcards
(113 cards)
1
Q
In common with all other organelles…
A
… the nucleus has it’s own
biochemical and molecular profile (repository for the genetic code)
2
Q
Traffic across …the nuclear envelope is
A
…highly regulated, involves the movement of nucleic acid (mRNA, tRNA, shRNA…) and specific proteins
3
Q
The nucleus is highly…
A
…. structured.
4
Q
What is th enucleus organised into?
A
eugenetic and heterogenetic
domains
5
Q
What is the nucleolus for?
A
a region for ribosome synthesis
6
Q
the nucleus is patitioned from the cytoplasm by a …
A
…. double membrane, known as the nuclear envelope
7
Q
the outer membrane is continuous with the …
A
… ER (endoplasmic reticulum).
8
Q
What are nuclear pores?
A
protein complexes (~100 proteins) >1250 kDa, tightly regulate
what goes in and out of nucleus
9
Q
how many nuclear pores per nucleus?
A
3000 - 4000 per nucleus.
10
Q
what is the inner membrane really important for?
A
nuclear structure and function
11
Q
Inner membrane associated proteins are essential for these processes, which include: ?
A
Lamin-B receptor (LBR)
Lamina-associated polypeptide-2 (LAP2)
Lamina-associated polypeptide-1 (LAP1)
Emerin
MAN1
12
Q
Inner membrane associated proteins all have an ….
A
N-term nucleoplasmictail, which is subject to posttranslational
modification (phosphorylation)
13
Q
What is lamin-B receptor involved in?
A
protein-protein interaction and exists in a large protein complex with:
LBR kinase
p18
p32/34
Lamin A
Lamin B
14
Q
Lamin-mesh of filaments (lamin A, B and C proteins) arrange on…
A
… the inside surface of the inner nuclear membrane (structural support)
15
Q
Lamins also interact with chromatin through…
A
… α-helical matrix attachment regions (MAR); that associate with 300 – 1000 bp (A-T) rich
genomic regions
16
Q
Lamin-mesh breaks apart during…
A
…nuclear disassembly
17
Q
Lamin-mesh breaks apart
during nuclear disassembly
(mitosis).
What is central to this process?
A
Phosphorylation of LBR by LBR kinase is central to this process
18
Q
LBR also interacts directly with …
A
chromatin proteins, such as HP1 (heterochromatin protein)
19
Q
Other inner membrane
proteins interact with
chroamtin…
A
Emerin, MAN1…etc
20
Q
Function of the inner membrane?
A
- Organises the nucleus; structural proteins and chromatin
- Also organises transcription sites (mRNA produced near membrane
pores; export much more efficient)
21
Q
Describe RFBP-RUSH complex.
A
- Ring finger binding protein (RFBP), interacts with RUSH with facilitates gene transcription through chromatin remodelling (interaction implicated in hormone regulation gene transcription)
- RUSH mediates switch of heterochromatin to euchromatin
22
Q
What does RUSH interact with?
A
RFBP (inner nuclear membrane protein)
23
Q
RUSH has a …
A
…ring finger domain, which binds DNA, RNA protein, and/or lipid
24
Q
RUSHinteracts with RFBP (inner nuclear membrane protein).
This interaction facilitates …
A
… the localisation of gene transcription with chromatin remodelling complex.
25
Positively charged histones (DNA binding proteins) form...
... strong interactions
with negatively charged
sugar-phosphate back bone
26
Histone acetylation removes...
... removes positive charge, releasing
DNA allowing transcription complex binding
27
DNA and histone methylation patterns also contribute to ...
...chromatin/nuclear architecture.
28
euchromatin transcription hotspots are ...
... highly organised.
29
DNA is not...
... ‘naked’ in the nucleus
30
How does dna exist in the nucleus?
exists as a nucleoprotein complex known as chromatin (histone / DNA complex = nucleosome)
31
What are the 5 different histone classes?
H1, H2a, H2b, H3 and H4 (in a nucleosome there is: 1H1, 2H2a, 2H2b, 2H3 and 2H4 = 9)
32
What do histones control?
Histones control two vital cell cycle events:
1) DNA replication during S phase
2) Chromatin condensation
(chromosomes)
33
There are several kinases responsible for phosphorylating.
What do these kinases do?
adding a phosphate group to histones.
34
Cyc;in dependatnt kinases (CDK) are able to...
... phosphorylate histone
35
Cyclin dependent kinase (CDK) are able to phosphorylate histone
e.g. Physarum polycephalum has four
phosphorylation sites on histone H1- Thr16, Thr136, Thr153 and Ser180
What are these sites known as?
mitosis associated phosphorylations
36
Thr136, Thr153 and Ser180 are of particular interest in this model, as phosphorylation at these sites promotes...
...nucleosome loosening, allowing access to DNA polymerase (S phase).
37
Removal of phosphaate from histones promotes...
...DNA condensation (chromosome formation)
38
WHats responsible for the removal of phosphate?
phosphatase.
39
It is the N-terminal histone tails which are the sites of ...
... post-translational modification (including phosphorylation)
40
How many acetyl groups can be added to h3 and h4?
up to four acetyl groups can be
added to H3 and H4 (histone acetylases or HAT enzymes) – loosen
nucleosome
41
Function of Methyl transferase methylate DNA, phosphatases and histone de
acetylases (HDAC) ?
remove phosphate and acetyl groups
(condensed DNA).
42
Important features of Stem Cells?
1) Unspecialised
2) Can differentiate (see semester 2)
3) Self-renewal (i.e. regenerate / lifelong)
43
At the top of the progeny map left is
the ‘stem cell’, with ...
... unlimited capacity to self-renew and produce ‘differentiated’
cells of various types
44
The ‘progenitor cells’, midway down have...
... limited capacity to self-renew and differentiate
45
Terminally differentiated cells do not ...
...self renew and do not differentiate
46
If stem cellscontain the same 46 chromosomes (genes) as terminally
differentiated cells, why do stem cells remain undifferentiated?
chromatin is organised differently in stem cells
47
DNAin the nucleus exists in ...
... a complex with Histone proteins
48
DNA wraps around a ...
... Histone
protein core
49
Histone proteins and DNA are ...
... biochemically modified
50
Biochemical modification alters the...
... wrap tightness
51
Loose wrap =
Euchromatin
52
Tight wrap =
Heterochromatin
53
What Charge is DNA?
Phosphate (PO4-) contains
oxygen, which creates negative
dipoles
54
Methyl groups (CH4) have no
oxygen and are...
... hydrophobic
55
Add methyl to DNA & Histone
tails creates...
... ‘sticky’ patches,
leading to heterochromatin
56
Add Acetate (C2H3O2-) to
histones creates...
... negatively
charged histones, which repel
negatively charged DNA leading
to euchromatin
57
Embryonic stem cells express a gene known as ...
...DNMT3L
58
What does DNMT3L code for?
one of the DNA methyltransferase chromatin remodelling enzyme
59
Embryonic stem cells express a gene known as DNMT3L which codes for one of the DNA methyltransferase chromatin remodelling enzyme.
The result is...
... euchromatin in regions of the genome that allow the expression of stem cell specific
genes (e.g. hTERT), whereas genes required for tissue specific differentiation are closed off.
This ensures that the stem cell remains unspecialised and is immortal.
60
Define Epigenetics
Epigenetics is the study of stable phenotypic changes that do not involve alterations in the DNA sequence
61
chromatin remodelling in stem
cells permits the expression of...
... stem cells specific genes, whilst switching off
tissue specific genes.
62
When we look at histone proteins in stem cells compared to terminally
differentiated cells, we see...
... chemical modifications unique to the stem
cell.
63
Histone protein 3 (H3) appears
to be really important in...
... maintaining the stem cell state
‘stemness’
64
Methyl groups (CH4) are added to...
... lysine residues (K) on the tails of H3
65
Methyl groups (CH4) are added to
lysine residues (K) on the tails of H3.
How many methyl groups can be added at any one time?
→up to three methyl groups can be added at any one time (hypermethylation)
66
In stem cells, H3 the lysine (K) at position 4 is hyper methylated (H3K4me3) as well as...
... H3 at lysine (K) at position 27 (H3K27me3).
67
Whats the function of H3K4me3 ?
This is seen in genes which are active
i.e. switches gene transcription on
68
What is the function of H3K27me3 ?
This inactivates gene transcription.
69
H3K4me3is observed in...
... the promotor region of several thousand
lineage specific genes in stem cells –yet these genes are not expressed.
70
H3K4me3is observed in the promotor region of several thousand lineage specific genesin stem cells –yet these genes are not expressed
H3K27me3 ...
... blocks these genes from being produced
71
This bivalent modificationof H3...
... poises the stem cell for differentiation
72
Genetic programming is different in stem cells – same genome, but the
chemical modifications of the DNA and Histones prevents...
... lineage specific gene expression
73
What is the nucleolus?
The nucleolus is a highly organised nuclear domain that is the site of ribosome synthesis
74
on what chromosomes are the p arms located?
13, 14, 15, 21 & 22.
75
Nucleolar organising region (NOR)
on p arms of chromosomes; 13,
14, 15, 21 & 22 ...
... aggregate, along with rRNA and ribosomal proteins (densely stained region within this
nucleus).
76
80% of RNA in a proliferating mammalian cell is...
... rRNA
77
15% of RNA is...
... tRNA
78
5% of RNA is ...
...mRNA (protein coding).
79
Ribosomes are...
... protein / rRNA complex; two
subunits 40s and 60s in higher eukaryotic cells
80
Ribosomal synthesis requires...
...RNA polymerases and associated proteins
81
Function of RNA polymerase I ?
transcribes rRNA
82
RNA polymerase II function ?
ribosomal protein transcription
83
~70 ribosomal proteins and 150 RNA / proteins that...
... interact transiently (during pre-rRNA editing)
84
46 protein subunits form a complex with ...
... 28s and 5.8s rRNAproducing the 60s ribosome subunit
85
Initiation factors (UBF, SL1 & TIFIA complex) bind to...
... upstream of rDNA gene, promoting RNA polymerase I binding
86
t-UTP sub-complex is necessary for...
... the transcription of the pre-rRNA molecule (unedited like the primary mRNA transcript)
87
Assembly of t-Utp sub-complex on nascent pre-rRNA promotes...
... the binding of other sub-complexes of proteins and snoRNA (short nucleolar RNA)
88
U3 snoRNA has ...
... complementary bases, and bind to the nascent pre-rRNA promoting rRNA folding and initiating pre-rRNA editing.
89
Additional snoRNA protein complexes bind, forming the...
... SSU processome, initiating A0 cleavage of pre-rRNA.
90
What does the SSU contain?
contains rRNA + small subunit proteins.
91
Describe progressive cleavage?
all sites: A1, A2 A3, B1, C2, D etc.. is required to
remove ITS1 and ITS2 and create the respective rRNA molecules that will complex with respective proteins to for the 40s and 60s ribosomal subunits.
92
Mature rRNA is complexed with...
...protein and exported to the cytoplasm
for ribosome construction
93
RNA exists as a...
... complex between nucleic acid and protein (RNP).
94
RNA is extensively...
... edited (5’ methyl cap added and 3’ poly-A
tail)
95
Splice factor proteins associate
with ...
... splice sites
96
Nuclear export factor (NXF1) and
nuclear export transporter (NXT1)
associate with...
... RNA and ancillary proteins
97
NXF1/NXT1 proteins interact with...
... FG hydrophobic cloud
98
NXF1/NXT1 proteins carry...
... RNP through the plasma membrane
99
RNA transport is...
... Ran-GTP independent
100
NXF1/NXT1 bind along the...
... length of the RNP → facilitate export
101
FG rich protein filaments extend
from...
... the FG core into the nucleoplasm (form nuclear basket) and into the cytoplasm
102
It is estimated that the nucleus
transports...
... 50 – 250 mRNA and 10 – 20 ribosomal RNP molecules per minute.
103
How mamny protein molecules transported per minute?
60,000
104
All nuclear proteins (histones, ribonuclear, transcription factors, lamins,
polymerase etc..) are...
... translated in the cytoplasm
105
Protein destined to reside in the nucleus contain ...
... nuclear localisation signal (NLS) peptide (signal patch): Pro-Lys-Lys-Lys-Arg-Lys-Val
106
Proteins transported in and out of the nucleus require...
... ancillary protein assistance (RAN, Nuclear transport receptor).
107
Whats the function of RAN ?
a molecular switch that alternates form
‘off’ to ‘on’ when bound to GDP or
GTP
108
Function of nuclear transport receptor?
binds to NLS of cargo protein (forms complex)
109
Ran is a G-protein (GTPase protein) involved in...
... nuclear transport
110
Ran when bound to GTP has a...
... particular conformation that recognises
the cargo protein.
111
Intrinsic GTPase activity of Ran converts...
GTP -> GDP (changes Ran conformation
112
Function of Guanine nucleotide exchange factor (GEF)?
protein that switch GDP → GTP in G-proteins
113
