Download The nucleus as an organized organelle

The nucleus as an organized organelle
• Individual chromosomes occupy distinct domains within the
interphase nucleus without overlapping other chromosomes
• The telomeres (ends of chromosomes), and condensed,
transcriptionally inactive regions (heterochromatin) are
attached to the nuclear envelope (via the lamina)
• Ribosomal RNA (rRNA) is synthesized in a site
corresponding to the nucleolus
• Pre-mRNAs are synthesized at 20-50 sites that appear as
speckles under fluorescent microscopy
• Rather than diffusing away from their sites of synthesis,
pre-mRNAs stream out towards the nuclear pores while
they are being processed into mature mRNAs
Spliced introns
Pre-mRNAs
Processed mRNAs
(b)
The nuclear matrix
• Treatment of eukaryotic nuclei with non-ionic detergents
and high salt, removes the histones and non-histone proteins
from chromatin, leaving a halo of DNA
• Digestion of DNA fibers with a nuclease (Dnase) leaves a
network of thin, protein containing fibers The nuclear
matrix
• Newly transcribed pre-mRNAs are associated with matrix
fibers, and treatment with an Rnase destroys the matrix. Id
nuclear matrix as a dynamic (moving) ribonucleoprotein
infrastructure
• The matrix is attached to the nuclear envelope, and may
represent an internal scaffolding for the nucleus
Packaging the genome: Chromatin structure
Levels of condensation
1. DNA wraps twice around a core
of histones (H2A, H2B, H3, H4),
forming nucleosomes
2. Nucleosome filaments (beadson-a-string) coil into “30 nm
fibers”
3. Fibers attach at intervals to a
protein anchor forming “loops”
approx. 50 kbp in length
4. During mitosis, the chromosome
condenses to ~1 cm DNA/µm
DNA double helix
(2 nm in diameter)
DNA
Nucleosome
core particle
Histones
(8 subunits)
Nucleosome filament
(10 nm in diameter)
30 nm fiber
Looped domains
Metaphase
chromosome
H1 histone
Replicating linear DNA: Telomerase
• The lagging strand of the
telomeres is synthesized by
telomerase (reverse
transcriptase)
• Telomerase activity is very
low in most cells. Shortening
of telomeres → aging
• Some cancer cells are
“immortal” (divide
indefinitely); contain high
telomerase activity → a
target for anticancer drugs
Telomerase
RNA
3' 5'
U
C
1
CCCCAACCCCAACCC
5'
U
A
A
AAA CCCCAAC
GGGGTTGGGGTTGGGGTTGGGGTTGGGG
3'
Elongation
CCCCAACCCCAACCC
5'
U
A
A
A AACCCCAAC
GGGGTTGGGGTTGGGGTTGGGGTTGGGGTTG
Translocation
UU
C
A
3'
3' 5'
U
C
3
CCCCAACCCCAACCC
C
A
3' 5'
U
C
2
UU
5'
U
A
A
A AACCCCAAC U U
GGGGTTGGGGTTGGGGTTGGGGTTGGGGTTG
3'
Elongation
3' 5'
U
C
4
CCCCAACCCCAACCC
5'
U
A
A
U
A AA CCCCAAC U
GGGGTTGGGGTTGGGGTTGGGGTTGGGGTTGGGGTTG
(b)
C
A
3'
C
A
Control of gene expression in eukaryotes
• Occurs at three primary levels:
1. Transcriptional-level control
2. Processing-level control
3. Translational-level control
• Transcriptional-level control
Steps in mRNA synthesis that are regulated:
1. Initiation: General factors (TFIID, TFIIB, TFIIH, TFIIE,
TFIIJ, etc..), accessory factors that bind to enhancer
sequences, and RNA polymerases (RNP I, II, and III)
Accessory factors bind to upstream (or downstream)
enhancers, and either increase (activation) or decrease
(repression) the rate for assembly of a PIC (pre-initiation
complex) on the promoter (TATA + initiator sequences)
Enhancer
DNA-binding
domains
Transcription
activating
domains
TFIIE
TFIIJ
TATA
TFIIB
TFIIH
TFIID
TFIIF
Initiator
RNAPII
Start site
DNA
Control of gene expression in eukaryotes
Many DNA-binding proteins
bind only to defined
sequences. They contain
domains e.g. bHLH, bZIP,
zinc-finger, etc.. with amino
acids that project into the
major or minor grooves,
where they hydrogen bond
with the DNA bases
Often, cooperative binding
through multiple contact
surfaces e.g. two subunits of a
dimer, increases selectivity.
3'
5'
108
Basic
region
124
166
Helix 1
Helix 2
137
146
Loop
5'
3'
Role of nucleosome positioning in
transcription
Nucleosome
• Nucleosomes may block the
access of TAFs to promoters
⇒ repression
• Nucleosome structure may
also aid cooperative
interactions between distant
sites ⇒ activation
• Histone:TAF interactions also
influence TAF binding to
promoters
Enhancer
Start site
Nucleosome
Transcription
factor
Promoter
(a)
Transcription
factor
Enhancer
Promoter
Nucleosome
(b)