Advanced Molecular Biology Flashcards
What is cell cycle?
Cell cycle is the ordered sequence of events that occur in a cell in
preparation for cell division
4 stages of the cell cycle
G1
S
G2
Mitosis
G1 phase
the cell increases in size
S phase
the cell copies its DNA
G2 phase
the cell prepares to divide
Interphase
It is the period between two cell divisions. Made of G1, S and G2 phase
Classification of proteins that play a role in stimulating cell division (4)
growth factors,
growth factor receptors,
signal transducers, and nuclear regulatory proteins (transcription factors)
G0
A deviation from the four cell cycle stages where a cell is not actively preparing to divide (It becomes quiescent)
Two ways cell cycle can be regulated
Regulation by external events such as growth hormone, cell size and neighboring cell death
Regulation by internal checkpoints located at the end of G1 and the S/mitosis transition. Also in metaphase.
What is a cell cycle checkpoint
A checkpoint is one of
several points in the eukaryotic cell cycle at which the progression of a
cell to the next stage in the cycle can be halted until conditions are
favourable.
What is the G1 checkpoint
The G1
checkpoint, also called the restriction
point (in yeast), is a point at which the cell irreversibly commits to the
cell division process. External influences, such as growth factors, play a
large role in carrying the cell past the G1
checkpoint.
Major role of the G2 checkpoint
the most important role of the G2
checkpoint is to
ensure that all of the chromosomes have been replicated and that the
replicated DNA is not damaged.
M checkpoint
The M checkpoint is also known as the spindle checkpoint,
because it determines whether all the sister chromatids are correctly
attached to the spindle microtubules
Two classes of cell cycle regulatory molecules
Positive
Negative
Positive regulatory cell cycle molecules
Cyclins
Cyclin dependent kinases
Cyclins regulate the cell cycle only when they are tightly bound to Cdks.
The 4 cyclin molecules
Cyclin D, E, A, B
How do cyclins and cdks work together
Cyclins flunctuate depending on the cell cycle stage and determine the cyclin-cdk complexed that form.
the cyclin-cdk complex must be phosporylated at certain parts to become active
CDKs
Cyclin-dependent kinases (Cdks) are
protein kinases that, when fully activated, can
phosphorylate and thus activate other proteins
that advance the cell cycle past a checkpoint.
To become fully activated, a Cdk must bind to a
cyclin protein and then be phosphorylated by
another kinase.
Cdk inhibitors
Molecules that prevent the full activation of Cdks
How can cdk inhibitor blocks on cdk be removed
Only when the specific event the inhibitor monitors has been completed
3 main negative regulatory molecules
Retinoblastoma protein, p53 and p21. All act primarily in the G1 checkpoint
Mode of action of the 3 main negative regulatory molecules
If damaged DNA is
detected, p53 halts the cell cycle and recruits enzymes to repair the DNA.
If the DNA cannot be repaired, p53 can trigger apoptosis, or cell suicide,
to prevent the duplication of damaged chromosomes.
As p53 levels rise,
the production of p21 is triggered. p21 enforces the halt in the cycle
dictated by p53 by binding to and inhibiting the activity of the Cdk/cyclin
complexes.
Rb exerts its regulatory influence on other positive regulator proteins.
Chiefly, Rb monitors cell size. In the active, dephosphorylated state, Rb
binds to proteins called transcription factors, most commonly, E2F. Transcription factors “turn on” specific genes, allowing the
production of proteins encoded by that gene. When Rb is bound to E2F,
production of proteins necessary for the G1
/S transition is blocked.
As the cell increases in size, Rb is slowly phosphorylated until it becomes
inactivated. Rb releases E2F, which can now turn on the gene that
produces the transition protein, and this particular block is remove
Proto-oncogenes
The genes that code for the positive cell cycle regulators. Proto-oncogenes are normal genes that, when
mutated in certain ways, become oncogenes, genes that cause a cell to
become cancerous
Oncogene
An oncogene is the altered form of any positive regulatory genes of the cell cycle in a way that it leads to an increase in the rate of cell cycle progression
Tumor suppressor genes
These are genes that code for negative regulatory molecules of the cell cycle. They are like car brakes, a malfunctioning brake would lead to a crash.
What is apoptosis?
Apoptosis (programmed cell death) is a genetically regulated
self-orchestrated naturally occurring cell death process that is active
during the course of development and induced during pathological
conditions for the overall benefit of the organism
What is necrosis
Unprogrammed/random cell death that causes inflammation
8 causes of cell death
ischemia,
hypoxia,
exposure to certain drugs and chemicals,
immune reactions, infectious agents,
high temperature,
radiation, and
various disease states
A hallmark feature of apoptosis
plasma membrane blebbing.
Apoptotic budding
separation of cell fragments into apoptotic bodies
Why does apoptosis not cause inflammation
apoptotic cells
or apoptotic bodies do not release cellular contents, are quickly
phagocytosed
4 biochemical features of apoptosis
Protein cleavage,
protein cross-linking, breakdown of DNA, and phagocytic recognition
What are caspases
Caspases are a family of molecules with proteolytic activity that can cleave
proteins at aspartic acid residues
Mention 10 caspases, grouping them into the 3 caspase groups
initiators (caspases-2, -8, -9, -10),
effectors or executioners
(caspases-3, -6, -7), and inflammatory caspases(caspases-1, -4, -5)
Phosphatidylserine
Normally facing inward in the cell’s plasma membrane. During apoptosis, phosphatidylserine is
oriented to the outside of the cell, where it is a well-known recognition
ligand for phagocytes.
Two other phagocytic markers
Proteins:
annexin I and calreticulin
Two stages of apoptosis
induction and execution
Three main apoptotic pathways
the intrinsic, or mitochondrial
pathway,
the extrinsic, or death receptor pathway
and Granzyme A and B pathways
Hallmark of extrinsic pathway
Activation of
transmembrane receptors, known as death receptors, by death ligands
What family do the death receptor/death domain belong
tumor necrosis factor (TNF) receptor superfamily, characterized by
the presence of extracellular cysteine–rich domains
5 death ligands/receptors
FasL/FasR,
TNF-a/TNFR1,
Apo3L/DR3,
Apo2L/DR4,
Apo2L/DR5
How is death receptor activation controlled
By inducible de novo expression of the
respective death ligands
Explain the extrinsic pathway using TNF-a and TNFR1
TNF-a binds to TNFR1, activating it’s death domain.
This leads to the recruiting cytosolic proteins like TNFR1 death domain protein- TRADD, Fas associated death domain protein- FADD and Receptor interacting protein- RIP.
These proteins interact with procaspase 8 which through autocatalytic activation becomes activated. This whole complex is the death inducing signalling complex- DISC, which then recruits and activates procaspase 3 to bring about the effector stage.
Perforin/Granzyme Pathway
Used by sensitized cytotoxic t-lymphocyte (CTL) cells and natural killer cells to clear harmful cells.
Involves the secretion of perforin which forms pores on the target cells. Then the CTL cells secrete cytoplasmic granules containing the proteases- granzyme A and B. These go in and ultimately lead to apoptosis. Granzyme B cleaves proteins at aspartate residues, activates procaspase 10 and cleaves factors such as inhibitor of
caspase-activated DNAse (ICAD). It can also directly activate the effector caspase- procaspase 3. It can also use the intrinsic pathway to amplify the death signal through the release of cytochrome c from the mitochondria.
Granzyme A plays a role in
CTL-induced apoptosis through the activation of a caspase-independent
pathway. Granzyme A causes DNA nicking by activating a specific tumor suppressor, and causing apoptotic DNA degradation.
Constitutive genes
Always turned on in the cell. Most important and control DNA replication, expression and repair
Regulated genes
Ones needed only occassionaly
Operon
A cluster of co-regulated genes including a promoter, operator which constitutes the regulatory part and the genes themselves which constitutes the coding part
Attenuation
A prokaryote specific regulatory process which involves the use of mRNA structure to stop both transcription and
translation depending on the concentration of an operon’s end-product enzymes.
3 regulatory methods of gene expression prokaryotes use
Repressor/Activator proteins
Attenuation
RNA polymerase structure
Default state of gene expression in eukaryotes
off
Role of histones in gene expression
Histones are proteins that are bound tightly to DNA in eukaryotic cells to form chromatin. They are dna silencers.
When the DNA is wound tightly around the histone, dna polymerase is unable to start transcription. However, histones are modifiable through specific chemical changes. It does this through the histone code. This
code includes modifications of the histones’ positively charged amino
acids to create some domains in which DNA is more open and others in
which it is very tightly bound up.
Methylation causes stricter silencing, acetylation causes histone unbinding.
Chromatin remodeling complexes
Complexes of proteins that use ATP to repackage DNA in more
open configurations
What is epigenetics
the study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself:
Transcription factors
a type of protein that regulates the synthesis of RNA from DNA during transcription by binding to specific DNA sequences. Could be activators or repressors
Two parts of a transcription factor
Effector and binding domain
Function of the effector domain of transcription factors
Recruiting RNA polymerase II
Two regions transcription factors bind to
Promoters and enhancers
4 ways eukaryotic gene expression can be regulated
Histone silencing
Transcription factors
Imprinting
X inactivation
Imprinting
involves the silencing of one of the two alleles of a gene for a
cell’s entire life span
Trp Operon regulation
When high levels of trp are present, the
repressor protein trpR binds the operator of the trp operon, preventing
continued expression of trp-synthesizing enzymes. However, trpR
requires the ligand tryptophan, the product of the enzymes encoded by
the operon, in order to bind the operator. It cannot bind the operator in
the absence of trp, thereby allowing continued expression of the trp
operon when the amino acid is needed
Trp attenuation procedure
Leader mRNA is the main player. The DNA region that codes for it is located between the operator and the coding region. This leader mRNA is able to change conformation depending by forming base pairings with itself. 2 possible pairings, one allows transcription and translation of the coding regions, the other stops it. It does this by using two tryptophan codons it has. When translation of the leader occurs, one of the two conformations form, depending on if the translating ribosomes stall at the tryptophan regions (meaning not enough tryptophan) or continues normally (meaning high tryp levels). If the ribosome stalls, the conformation for continued transcription and translation is formed, if not then the attenuator stops transcription.tryptophan is readily added, it signifies excess in the cell. As such the mRNA interacts with itself in a way that it stops further transcription and translation. If the translation stalls at the tryptophan region, it means tryptophan is not readily available and as such, the structure formed allows the continuous transcription and translation.
Induction in gene expression
This brief delay from
basal expression to induced expression
Lac operon regulation process
Lac repressor protein is always bound to the promoter of the lac operon in the absence of lactose to prevent synthesis of enzymes that break down lactose (Note: It’s not perfect so there is still a basal transcription, hence production of these enzymes). When lactose is present, allolactose, a molecule formed from lactose binds to the repressor and causes it to separate from the promoter, allowing transcription. The amount of lac proteins produced is still minimal and depends on positive control for the levels to rise.
For positive control, a particular protein CAP (catabolite activator protein) is necessary to bind to the activator binding site for increased lac gene transcription. But to be activated, it has to be bound to cAMP. As glucose levels drop, cAMP levels rise, causing it to bind to CAP, ultimately leading to the increased production of the lac genes mRNA
2 levels of eukaryotic gene expression regulation
Control from the amount of mRNA produced (Transcriptional)
control via post-transcriptional mechanisms that regulate translation. (Translational)
The most important
structural difference between eukaryotic and prokaryotic DNA is
the
formation of chromatin in eukaryotes