Lecture 15 Flashcards
The human genomic DNA is in how many chromosomes?
46 chromosomes or 23 pairs
What does packaging DNA do?
Protects it from damage and contributes to regulation of replication & transcription
Why is it important to package DNA so small?
To be able to fit into the nucelus
What stages are in interphase?
- G1, S, G2
- G0 for non-dividing cells
During interphase, what form does DNA take?
DNA is present as chromatin
What is the appearance of chromatin?
- Appears amorphous and randomly distributed
- It is “uncondensed”
What does the S phase of interphase stand for?
Synthesis
What happens to the chromosomes during S phase?
Each chromosome is replicated, resulting 2 sister chromatids that remain associated
What are the stages of mitosis?
Prophase, metaphase, anaphase, telophase
What occurs during prophase of mitosis?
The chromosomes become more compact, forming condensed chrmosomes
How does the shape of chromosomes change after cell division?
Once cell division is complete the chromosomes again become uncondensed and the cycle repeats.
Is chromosome or chromatin more condensed?
DNA is more condensed in chromosomes than in chromatin but chromatin DNA is still condensed and has multiple levels of organization.
What happens in interphase of mitosis?
DNA replicates
What happens in prophase of mitosis?
- Chromosomes condense, the spindle forms, and the nuclear envelope disintegrates
- Each chromosome has two chromatids
What happens in metaphase of mitosis?
Each chromosome aligns independently at the metaphase plate
What happens in anaphase of mitosis?
Chromatids separate
What happens in telophase and cytokinesis of mitosis?
The nuclear envelope reforms and the cell now divides (cytokinesis)
After cell division a typical eukaryotic cell contains how many copies of each chromosome
- 2 copies of each chromosome (is diploid)
When does the synthesis of histones occur?
Late in the 1st “gap phase” (G1)
Fxn of histones?
Package and order DNA into structured units
During histone synthesis the cell enters what phase in order for what process to begin?
The cell enters S phase and DNA replication begins
Which strand(s) are histones and non-histone proteins deposited onto to produce chromatin?
Both the template and daughter DNA molecules to produce chromatin
When DNA replication is complete which phase does the cell enter?
2nd gap phase, G2
At the end of G2 the cell is ready to enter what?
Mitosis where it will divide, distributing one copy of each chromosome to each daughter cell
What does chromatin consist of?
DNA + histones + non-histone proteins
What is heterochromatin?
Condensed chromosome
What is the nucleus?
Membrane-bound organelle in eukaryotic cells that contains the genetic material
What is on the nuclear envelope’s inner and outer membrane?
Nuclear pores
What are nuclear pores
Elaborate gates that control transport of small and large molecules to/from the nucleus
The nuclear membrane is continuous with ___
The endoplasmic reticulum
What is the nuclear lamina
The inner surface of the nuclear envelope
What is the nuclear lamina lined with?
Proteins called nuclear lamins
What do nuclear lamins form?
Intermediate filaments that also extend across the cytoplasm to provide structural support
How do chromatin and nuclear RNAs attach to the nuclear membrane?
Via the lamins
What is the fxn of endoplasmic reticulum?
Shares nuclear membrane
What is the nuclear pore complex?
Highly complex proteinaceous pore that regulates the entry/exit of proteins, and exit of mRNA (proteins are made in the cytoplasm from mRNA – some are transported back into the nucleus)
Fxn of nuclear lamina
Provides structural rigidity to nucleus and, site of attachment for chromatin
Fxn of nuclear matrix
Diffuse nuclear scaffold consisting of proteins that attach chromosomes to the nuclear envelope or other structures in the nucleus
Fxn of nucleolus?
A ribosome-producing sub-compartment of the nucleus
What is nucleoplasm?
Chromatin/chromosome-containing region
What is chromatin?
Chromosomal material – protein-DNA complex
What does chromatin consist of?
fibres containing ~equal masses of DNA and protein, along with a small amount of RNA
DNA in chromatin is tightly associated with what proteins? Their fxn?
- Histones
- Protect and package the DNA and regulate replication and transcription by limiting its access
What is the 1st lvl of chromatin packaging?
Nucleosomes
What occurs in 1st lvl of chromatin packing?
- Histones package and order the DNA into structured units called nucleosomes
- Nucleosomes are packaged into higher order structure
- Chromatin also contains non-histone protein
What are nucleosomes?
Fundamental unit of chromatin aka beads-on-a-strings
What are the major protein component of chromatin
Histones
How does the charge of histones affect DNA?
Histones are small, very basic proteins that contain ~25% lysine’s and arginines to neutralize the highly charged DNA
What does the assembly of histones with DNA require?
Molecular chaperones
What are the 5 types of histones?
- H2A
- H2B
- H3
- H4
- H1
What are the core histones? Purpose?
- H2A, H2B, H3, H4
- 2 copies of each form an octamer (8 subunits). The DNA is wrapped around the histone core to form the nucleosome
What is the fxn of H1
Clamps DNA wrapped around nucleosome
How do histones compare between species?
Histones are very highly conserved. For example, H4 is 98% identical between cows and peas
The DNA in the nucleosome is ~2 loops
of ___ supercoil
secured to the histone core by histone
protein H1.
- Negative supercoil (under wound) (note: negative supercoil
even though the coil is left-handed.)
What is the linker DNA?
DNA between histone wrapped nucleosome
What is link DNA digested by?
Nuclease
What does a nucleosome include?
~200 nucleotide pairs of DNA and core histones
How are nucleosomes dissociated?
- Dissociation with high concentration of salt
- This disrupts the electrostatic interactions between the histone proteins and the DNA
What is the second level of chromatin organization?
- 11nm chromatin fibers coil to form 30nm chromatin fibers or solenoids
- provides ~100-fold compaction of DNA
Chromatin in interphase and non-dividing cells are in what level of chromatin organization?
- 2nd level of chromatin organization (30nm fiber)
What is the role of histone H1 in compaction to the 30nm fiber?
H1 holds the DNA on the histone core and is necessary for the formation of the 30nm fiber.
Where is H1 located in the 30nm fiber?
- the interior
RNA synthesis (transcription) occurs during interphase when chromosomes are in the 30nm fibers. Protein complexes must associate with the DNA and scan along it, opening up the duplex to synthesize daughter strands & make RNA. Thus what happen for this to occur?
- Regions of tightly packed DNA in the 30nm fiber must become partially dissociated to be accessible to the DNA replication and transcription machinery… this is accomplished using histone modifying enzymes and chromatin remodelling proteins
When the 30nm fiber further compacts to form condensed chromosomes what is the overall compaction?
10,000 fold
What is the nuclear scaffold made of?
- Mainly H1
- Topo II
Why is Topo II needed in the nuclear scaffold?
Required to supercoil the DNA during chromatin assembly
Further looping/coiling of solenoid DNA into highly compacted, transcriptional silent form of chromatin called?
Heterochromatin
When does heterochromatin formation occur?
During mitosis
Histone 2A and 2B form a ____ dimer through an interaction known as the ___
- Head to tail dimer
- Handshake
2 H3/H4 dimers associate to form what?
A tetramer
2 H3/H4 dimers associate with 2 H2A/H2B to form what?
An octamer
Nucleosome core particle is consisted of what two things?
Histone core + 146 bp DNA
DNA wraps tightly around histone octamer with exactly how many and what type of turn?
1.65 turns of negative supercoil
In nucleosomes, what do (positively charged) histones interact with in DNA that explains the histone’s lack of sequence specificity?
Histones interact primarily with the sugar-phosphate backbones
What interactions does the exposed DNA surfaces in nucleosomes allow for?
Interactions between DNA surfaces and DNA binding proteins
How much of the DNA surface is exposed and available to interact with the DNA binding proteins in the 11 nm fiber?
70%
What protrudes from the octamers (core histone)?
N-terminal tails (rmb histone core is an octamer made from H2A, H2B etc… so the tails are H2A tail, H2B tail etc.)
What is the charge on the N-terminal tails of the histone protein and why?
Positively charged due to many Lys and Arg residues
How does the N-terminal histone tails help mediate assembly of 30nm fiber?
Histone tails pack nucleosomes into 30nm fiber cause positively charged N-termini bind negatively charged sugar-phosphate backbone on DNA of neighbouring nucleosomes
What holds the 30nm fiber?
- Histone H1 proteins, which “pull” the nucleosomes together
- N-terminal tails
Where can gene transcription (RNA synthesis) not occur?
In the solenoid
What are the 3 possible covalent modifications on histone tails?
- Phosphorylation of series (-PO4)
- Irreversible methylation of lysine’s (-CH3)
- Reversible acetylation of lysine’s (C=O - CH3)
Histones are modified in the assembled nucleosomes in order to regulate what two things?
- Chromosome structure (compaction)
- Gene activity (i.e. transcription)
Do nuclear enzymes add or remove modifications?
They do both
What is histone acetylation?
Reversible modification of lysine’s in the N-terminal regions of the core histones
Results from histone acetylation?
- Loss of positive charge reduces binding to DNA and destabilizes chromatin
- Acetylated regions attract proteins that can either cause further compaction or can facilitate access to DNA
What reverses histone acetylation?
Deacetylases
Where do covalent modifications occur in histones?
On specific residues in the N-terminus of the histones
What is the patterns of modification called?
Histone code
What recognizes the histone code?
Enzymes that alter the structure of chromatin e.g. modifications associated with transcriptional activation would be recognized by enzymes that make the chromatin more accessible to the transcriptionally machine, same for replicate machinery
What does chromatin remodelling alter?
- The accessibility of DNA to DNA-binding proteins
- change nucleosome structure to change the positionsof nucleosomes on the DNA (translocation) orto change the structure of DNA within the nucleosomes so that DNA is not as tightly associated with the histone core
How can chromatin become “remodelled” (2 things)
- enzymes that covalently modify histones e.g. histone acetyltransferases, deacetylases etc.
- the work of chromatin remodelling complexes
What do chromatin remodelling complexes do?
Work to inc or dec its accessibility to proteins in the cell, particularly those involved in gene expression, replication and repair, thus chromatin structure is dynamic
Do chromatin remodeling complexes require energy?
Yes they hydrolyze ATP to remodel or translocate nucleosomes
How is DNA replication during interphase done
As the replication fork approaches a stretch of chromatin, nucleosomes on the parental duplex are displaced and new ones (containing both re-cycled and new histones) are re-formed on the newly synthesized daughter duplexes.
What are 5 non-histone DNA binding proteins used during replication?
DNA polymerases, ssDNA binding proteins, Topo I and II, helicase, primase etc.
What are 2 non-histone DNA binding proteins used during transcription?
RNA polymerases, transcription factors (activators and suppressors)
What are 2 non-histone DNA binding proteins used during RNA processing?
factors for ribosomal assembly (ribosomal proteins), nuclear RNPs (ribonucleoproteins) involved in mRNA processing
What are 4 other non-histone DNA binding proteins?
Telomere associated proteins, centromere associated proteins, scaffolding proteins, recombination proteins
Chromosomes must be replicated, and the sister chromatids separated to allow one complete copy to be passed on to each daughter cell. This process is controlled by what three DNA sequences within the chromosome? All three sequences are necessary for a functional chromosome.
the replication origin (many), the centromere and the telomere (two)
What is the replication origin?
- the location at which DNA duplication beings
- eukaryotic chromosomes contain many origins or replication to ensure that the entire chromosome can be replicated rapidly
What is centromere?
- attachment site for mitotic spindle (via a protein complex called a kinetochore)
- allows for one copy of each duplicated and condensed chromosome to be pulled to each daughter cell during cell division
What is a telomere?
- formed at the end of chromosomes
- contain sequence repeats that enable the ends of chromosomes to be efficiently replicated
- protect the ends of chromosomes from nucleases and from being recognized as “breaks” in need of repair
