Unit 3 : Nuclear Structure & Function Flashcards
Nuclear Envelope
double membrane shrouding contents of nucleus
inner + outer membrane with perinuclear space between
outer membrane continuous with ER
perinuclear space continuous with ER lumen
cytoplasm and nucleoplasm connected through nuclear pores
Nuclear Lamina
directly under inner membrane of envelope
meshwork of intermediate filaments
support nuclear envelope - keeps nucleus from being torn apart during normal cell processes
Chromatin
Interphase DNA
attached to nuclear envelope via lamina
During mitosis
nuclear lamina breaks down nuclear envelope so chromosomes released
>nuclear laminas phosphorylated
> conformational change in lamins and causes
destabilization of the nuclear lamina, which
results in its breakdown
Interphase Chromatin level of stabilization
Highly organized due to presence of sub-cellular regions in nucleus
Nucleolus
Prominent sub-cellular regions in the nucleus
visible by TEM
Function of nucleolus
synthesize all of the ribosomes the cell needs to continue to function
Ribosomes
ribonucleoproteins
made up of proteins + RNA
Nucleolus organizer regions
rDNA regions
regions around where nucleolus forms
Nuclear Pores
control the transport of macromolecules in and out of the nucleus
only way to access the nucleus
Nuclear Import/ Export happens via the Nuclear Pores
2 possible mechanisms depending on size of molecule
1) Very small molecules - diffuse through the center
of the pore without help
2) Molecules that are larger than the diffusion limit
- require the input of energy to facilitate
transport ( GTP used )
Diameter of nuclear pores
passage of material by free diffusion is about 9 nm
molecules larger than 9nm strictly controlled
molecules that are too big are not allowed to pass
What goes in nuclear pores ?
Histone proteins
Polymerases
Transcription factors
Ribosomal proteins
What goes out of nuclear pores ?
Ribosomal subunits
mRNA - protein complexes
fully processed spliced transcripts
Protein targeting to nucleus
there is a specific signal that is used by the cell to identify what can enter the nucleus, what can exit
proteins that have business in the nucleus must contain within their amino acid sequences signals that are recognized by the system
All proteins that must enter a membrane-bound organelle will be specifically targeted there by some mechanism encoded in the primary sequence of the protein
Targeting signals
First - every protein that is sent (targeted) to a specific site within the cell must have a destination-specific code associated with it
Second - there must be some sort of specific receptor for destination
differences in the final destination of proteins
consequence of targeting signals contained within the primary amino acid sequence of the protein itself
consensus sequences
they show the most common amino acid sequences that are used for a specific type of protein targeting
1 ) If one lines up the amino acid sequences of a number of proteins that are targeted to a particular organelle - they all contain this sequence or something very similar to it
2 ) some amount of variation in the signal that is used to enter a specific organelle but we are disregarding this
Import into the Nucleus
Targeting Signal: KKKRK
or
‘-Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val-‘
KKKRK
internal targeting sequence
must also be on the surface of the 3D protein
proteins are imported into the nucleus after translation, in a folded state
occurrence of this targeting sequence in a protein is said to be both necessary and sufficient
Steps of Nuclear Import
1 ) NLS is first recognized on the protein to be imported - KKKRK
2 ) NLS region on the surface of the nuclear protein binds to a soluble cytosolic NIR and forms a protein-receptor complex
3 ) The protein-receptor complex binds to the cytosolic fibril of the nuclear pore –> GTP-driven reaction results in a change in the configuration of the pore –> translocation (movement) of the protein complex through the pore and into the nucleus.
4 ) Once inside the nucleus, the nuclear import receptor dissociates from the nuclear protein and returned to the cytosol.
5 ) NLS remains part of the nuclear protein
Export of Proteins from the Nucleus
protein contains a nuclear export signal that binds with a receptor –> binds to the nuclear pore –> nuclear export receptor and the protein dissociate after transport –> export of ribosomal subunits through nuclear pores works through a more complex similar process
DNA packing in Interphase Nucleus
DNA will be packed up so tightly that it cannot be accessed and read
When gene expression is required - specific regions of the packed DNA will be loosened so that transcription factors and other expression machinery can bind to the DNA and transcribe it
Packed DNA called
chromatin
30nm fiber - average diameter of chromatin measured by TEM
Chromatin is formed from
DNA and Histones
Chromatin can be remodeled and regulated by
non-histone chromatin-associated proteins
Non-histone chromatin-associated proteins
heterogeneous group that interact with DNA and/or histones
Histones
set of basic proteins that interact strongly with DNA
overall + charge due to basic amino acid side
chains
Core histones
H2A H2B H3 H4
interact strongly with each other and with DNA to form nucleosome
H1
unique histone that binds to outside of nucleosome
helps pack nucleosomes together to pack DNA tightly
Nucleosome core particle
DNA + core histones
Linker DNA
DNA between each nucleosome core particle
Nucleosome
Nucleosome core particle + Linker DNA
histone tails
important in the regulation of chromosome structure including euchromatin to heterochromatin
First level of packing
Beads-on-a-string
Beads-on-a-string
call it the 10 or 11nm fiber
it is not really seen in a live cell
result of experimental manipulation to remove the H1 histone and unpack the DNA
Second level of Packing
Chromatin
Chromatin
11nm fiber is further condensed into the 30nm fiber through interaction with Histone H1
30nm chromatin fiber is the naturally occurring level organization of chromatin in the interphase nucleus
Upper levels of Chromatin packing
non-histone scaffolding proteins further pack 30nm in interphase
help make large chromosomal loops of DNA with genes exposed at ends of loops
Separating DNA and histone proteins
ionic salt washes
ionic salt washes
dissociates chromatin due to non-covalent interactions between DNA and proteins
Summary of Nuclease Digestion Experiment
1 ) Lightly digest chromatin with nuclease
2) Remove all associated proteins
3) Separate DNA molecules by size using gel electrophoresis
key idea is to lightly digest chromatin with an enzyme that selectively digests
exposed DNA only
enzyme that selectively digests is
micrococcal nuclease
Key idea
micrococcal nuclease can be used to determine what DNA is tightly bound to proteins thus ‘protected’ from cutting by nuclease
All unprotected DNA will eventually be destroyed if the nuclease is left to digest for long enough
Key assumptions of this experiment
If there is a repeating protein structure that associates with the DNA there will be protected pieces of DNA left after nuclease digestion
These pieces of DNA will have a standard size if they are bound to the same type of structural complex
Gel Electrophoresis of DNA fragments
Used to separate DNA (or RNA)
Gel made of agarose
DNA already - charged so no SDS required
result is a banding pattern on a gel that tells use about relative size and abundance of each DNA fragment
SDS-PAGE
Used to separate proteins.
gel is made of polyacrylamide
Prior to electrophoresis - proteins must be pre-treated with SDS and heat to denature and coat them with a uniform negative charge
End result is a banding pattern on a gel that tells use about relative size and abundance of each protein
Transcriptional control
when and how often genes are transcribed
RNA processing control
which combinations of introns/exons are produced so different proteins can be made from the same gene
Once the processed mRNA leaves the nucleus
the cell continues to regulate the gene products by controlling
when and how translation happens
when the mRNA is degraded
what kinds of post-translational modifications take place
when the protein is tagged for destruction
Control of Chromatin Structure
Controls Access to Genes
Interphase chromatin / 30nm divided into
Constitutive Heterochromatin
Facultative Heterochromatin
Constitutive Heterochromatin
heterochromatin is always condensed
happens in structural areas centromeres and telomeres
no genes to be found in areas with constitutive heterochromatin
Facultative Heterochromatin
heterochromatin that is not always condensed
Genes within facultative heterochromatin have been shut down temporarily by restricting access to the DNA - very important way that the cell controls gene transcription
Mutations in the genes that help regions of the chromosome transition between euchromatin and heterochromatin can be quite serious, and often result in cancer
Modification of Chromatin & Histones
Controls Access to Genes in DNA
allow or restrict access to genes controlled by
1 ) Histone modifying enzymes
2 ) Chromatin remodeling complexes
Histone Modifying Enzymes
chemically alter the histones of the nucleosome core
tails are modified by the addition of a variety of chemical functional groups the most common modifications include acetylation (Ac), methylation (M) and/or phosphorylation (P)
what determines what happens to a particular stretch of chromatin at any given time
pattern of these modifications
Chromatin Remodeling Complexes
Modification of the histone tails commonly results in formation of specific binding sites for enzymes which then bind to the entire nucleosome complex and ‘shift’ the DNA that is wrapped around it so DNA can be re-positioned - process is absolutely essential for genes to be exposed and expressed
evidence to suggest that during mitosis many of the chromatin remodeling complexes are
inactivated - so heterochromatin can be formed efficiently without any chance DNA loosens again
Transcription Factors Control Transcription at the Gene Level
required to allow the RNA polymerase to bind to the DNA for initiation
can enhance or inhibit transcription
The Structure of a Eukaryotic Gene - Review
The coding strand
The template strand
key DNA sequences in this transcription unit that should be highlighted
5' flanking sequence Enhancer region Promoter region Transcription start site Transcription stop site 3' flanking sequence Translation start site Translation stop sight
Transcription Factors control
when and how Transcription Happens
Activators (protein) bind
Enhancer regions
Repressors (protein) bind
Suppressor regions
Co-factors
work together with other regulators to change the transcriptional response
Chromatin remodeling complexes
bind to nucleosomes and help promote the transition between euchromatin & heterochromatin
Transcription Factors and Chromatin Remodeling Complexes Work Together to
control Eukaryotic Gene Expression
Fig 8-11
On the left, the activator acts as a binding site for a histone modifying enzyme
On the right, the activator creates a binding site for a chromatin remodeling complex
General Principles of Transcript Processing
All transcripts are processed (mRNA, rRNA, tRNA) in the nucleus before they are transported to the cytoplasm
Processing is carried out by proteins (and RNA) that bind to and modify the transcripts
Virtually all processing signals are encoded into the primary sequence of the RNA transcripts themselves
Processing may include any of the following modifications:
1) Addition of sequences (e.g. 5’ cap and polyA tail in mRNA)
2) Cleavage of the transcript into several pieces (rRNA)
3) Removal of some sequences (all classes of RNA)
4) Splicing (i.e. removal of sequences by cleavage, followed be re-joining of remaining RNA fragments back together)
three different RNA polymerases in the nucleus
Polymerase I
Polymerase II
Polymerase III
3 major processing events that happen with mRNA
RNA capping
Polyadenylation
Splicing
snRNPs
small nuclear RiboNucleoProteins
enzymes that contain a small RNA molecule that is complementary to the recognition sequences in the RNA transcript
come together to form a large complex called the spliceosome
Summary of the Process of RNA Splicing
the formation of the spliceosome begins when the RNA portions of the snRNPs recognize the intron/exon junctions and base pair with them
snRNPs arrive and interact to bring them together and form the complete spliceosome
The second cut in the transcript occurs at the right edge of the intron and the two exons are joined together
promoter choice
some genes will have two or even more promoters, each of which leads to the production of a different initial exon
trans-splicing
exons from 2 separate gene transcripts are spliced together to produce a completely new mRNA
