Unit 3 - Nuclear structure and function

3.1 Nuclear structure and protein import

Describe the structure of an interphase nucleus, and recognize structural elements in different kinds of microscopy.

Overview of nucleus:

Nuclear envelope

  • Double membrane surrounding nucleus

  • Inner membrane → perinuclear space → outer membrane (also continuous with the ER)

Nuclear lamina

  • Lies under the inner membrane of the nuclear envelope

  • Formed from a mix of intermediate filaments that support the nuclear envelope

  • Help disassemble and reassemble nuclear envelope during cell division

Nucleolus

  • Synthesize ribosomes the cell needs

  • Nucleolus organizer regions

DNA - euchromatin & heterochromatin (condensed)

Interphase:

  • Interphase DNA (chromatin) is attached to the nuclear envelope through the nuclear lamina

  • In mitosis, nuclear lamina breakdown nuclear envelope for chromosomes to be released and spindle can form

  • Nuclear lamina are phosphorylated → conformation change in lamina → destabilize and breakdown nuclear lamina → nuclear envelope breaks down

  • When mitosis is over, the cell removes the phosphate groups from the lamina which reforms the nuclear lamina → nuclear envelope is reformed

Describe the nuclear pore complex (NPC) and explain the different types of nuclear transport mechanisms.

  • Big protein complex on the membrane of the nucleus

  • Cytoplasmic filaments sticking out

  • NPC has channel nucleoporins which is a messy tangle

  • Nuclear pore control transport of macromolecules in and out of the nucleus

  • Two transportation mechanisms depending on the size of the molecule

    • 1. Very small polar molecules (water, ions, ATP, GTP)

      • Diffuse through the centre of the pore without help

    • 2. Molecules that are larger than the diffusion limit

      • Requires the pore to stretch to accommodate them

      • Uses GTP

      • If bigger than 9 nm, they are controlled and if they are too big, they can’t pass

Explain how proteins are transported into and out of the nucleus, including the roles of the Nuclear Localization Signal (NLS), nuclear transport receptors, and the NPC itself.

  • You need the protein that is being transported (signal sequence = KKKRK), the receptor protein, energy (GTP), and the nuclear pore complex

  • Specific signal used by the cell to identify what can enter the nucleus and what can exit

    • Recognizes signals sequence and binds cargo protein

  • All proteins have a membrane-bound organelle will be specifically targeting….

  • Transported proteins contains a targeting signal in its primary amino acid sequence (KKKRK)

  • Every protein that is sent to a site within a cell needs a destination-specific code

  • And, there needs to be a specific receptor for destination

  • Import:

    • Nuclear localization signal (NLS)

    • Nuclear import receptor (NIR)

  • Export:

    • Nuclear export signal (NES)

    • Nuclear export receptor (NER)

Protein targeting:

  • Targeting signals are encoded within proteins

  • Targeting signals direct the protein to a specific organelle

  • Targeting signal must be present for protein to leave the cytosol compartment

Nuclear import:

  1. NLS is recognized on the protein to be imported with KKKRK

    • NLS needs to be accessible on the surface of the 3D protein to be recognized. Found near the N-terminus

  2. NLS region of the nuclear protein bind to a soluble cytosolic nuclear import receptor protein and from a protein-receptor complex

  3. The protein-receptor complex bind to the cytosolic fibrillation of the nuclear pore. Stretches pore and protein complex moves through the pore and into the nucleus

  4. Inside, the nuclear import receptor dissociates from the nuclear protein and returned to the cytosolic

  5. NLS remains apart of the nuclear protein

Import (cytoplasm → nucleus)

  1. Protein cargo has nuclear localization signal (KKKRK)

  2. Nuclear import receptor (importin) recognizes NLS and binds

  3. The receptor binds to specific nuclear pore

  4. Tranportation occurs

  5. RanGTP binds to the importin which changes the conformation and let goes of the cargo protein

  6. RanGTP-importin complex goes back out

  7. RanGTP is hydrolyzed to RanGDP and dissociates from importin

Nuclear export:

  1. The protein has a nuclear export signal that binds with a receptor and then binds to the nuclear pore

  2. Mature RNA ready of export must be bound by proteins

    • NES is an amino acid signal meaning that RNA can’t possess it

  3. After transporting the protein, the nuclear export receptor dissociates and protein split apart

  1. Nuclear export receptor (exportin) is a protein inside the nucleus that mediates transport (export)

  2. Exportin binds to RanGTP

  3. Nuclear export signal on cargo protein binds to the two proteins

  4. Goes through the pore

  5. In the cytosol, GTP hydrolyzes ranGTP to ranGDP which detaches from the two other proteins

  6. Exportin unbinds and goes back to the nucleus

  7. Cargo protein is now in the cytosol

Discuss how the primary sequence of a protein contains all of the information to determine whether a protein is imported into the nucleus, using experimental evidence from fluorescence microscopy

Loss of function experiments:

  • Remove the thing from the system and see what happens

  • Shows if the thing you removed in necessary

Gain of function experiments:

  • Add a component that isn’t normally present and see what happens

  • Shows if the thing you add is sufficient

Analyze experimental evidence from fluorescence microscopy and explain how it provides evidence that the primary sequence of a protein contains all of the necessary information to determine whether a protein is imported into the nucleus.

3.2 Chromatin & chromosomes

Discuss how proteins and DNA interact to form chromosomes, starting with the 2nm naked DNA molecule to the structure of interphase chromatin, including euchromatin, heterochromatin, and chromosomal loops.

  • Chromatin needs to be functionally organized

    • Tightly packed chromatin (30 nm) during interphase

    • Chromatin is made from DNA and histone proteins. It can be removed and regulated by non-histone chromatin proteins

    • Heterochromatin: densely staining region, concentrated around centromere and telomeres of chromosomes for protection, no transcription

    • Euchromatin: lightly stained regions, genes are actively being transcribed

  • DNA is compacted to fit

    • Packing DNA three levels

    • 1. Naked DNA

    • 2. Beads on a string form of DNA (10 or 11 nm)

      • Can’t be seen in a live cell

    • 3. Interphase chromatin (30 nm)

      • Has histone H1 binds DNA

      • Native level organization of chromatin in the interphase nucleus

      • These create chromosomal loops of DNA

  • Need to be accurately divide chromosomes for cell division

    • Loops of 30 nm chromatin formed by non-histone chromatin proteins that form a scaffold

  • DNA needs to be accessible

Explain how the primary, secondary, tertiary and quaternary levels of histone structure contribute to nucleosome assembly.

Interpret experimental results providing quantitative information about the spacing of nucleosomes and the amount of DNA associated with these structures.

3.3 Regulation of gene expression

Discuss the different types of DNA and histone modifications and their roles in the regulation of gene expression.

Types of chromatin:

  1. Euchromatin - Chromatin that is transcriptionally active

  2. Heterochromatin - Transcriptionally inactive DNA that has been tightly condensed to prohibit access

    a. Constitutive heterochromatin

    • Always condensed in structural areas (centromeres, telomeres)

    • No genes to be found

    b. Facultative heterochromatin

    • Not always condensed

    • Genes within facultative heterochromatin have been shut down temporarily by restricting access to DNA

    • Important way that the cell controls gene transcription

    • Mutations in the genes that help regions of chromosome transition between euchromatin, and heterochromatin are serious

Modifications:

  1. Histone modifying enzymes

    • Chemically alter the histones of the nucleosome core

    • The 8 core histones have a short tail that can be accessed by histone modifying enzyme inside the nucleus. These tails are modified

    • Acetylation

    • Methylation

    • Phosphorylation

    • The pattern of the modifications determine what happen to a particular stretch of chromatin

    • The modifications can act as binding sites for specific non-histone proteins

  2. Chromatin remodeling complexes controls access to genes

    • Modification of histone tails result in forming specific binding sites for the enzymes which binds to the entire nucleosome complex and shift the DNA that is wrapped around it (DNA can be repositions)

    • Essential process for genes to be exposed and expressed

    • During mitosis, chromatin remodeling complexes are inactivated so heterochromatin can be formed efficiently without the chance of DNA becoming loose again

  3. Transcription regulators binding

slides:

Transcription regulation

  • General transcription factors bind to promoter to help recruit RNA polymerase

  • Other PRO being to regulatory regions to promote or repress transcription

Distinguish between the different types of transcription factors/regulators (e.g. basal, activators and repressors) and explain how their interaction with specific regulatory regions of DNA regulate transcription.

Discuss how mRNA processing events (e.g. cap, tail) can regulate overall gene expression.

Discuss the mechanism and specificity of mRNA splicing events and explain how alternative splicing increases the diversity of protein products encoded from a single gene.

Illustrate the importance and specificity of macromolecular interactions involved in regulation of gene expression by interpreting experimental evidence in eukaryotes.