Cell Regulation
Describe the consequences of cell cycle checkpoint failures in mitosis and meiosis (Ask Akaash for a hint if you need it since its the $100)
Example: viruses induce cell proliferation
Viral Rb-bonding proteins bond to Rb so E2F-1 gets released, stimulating S-phase proteins
Increases p53 activity but then there are proteins made that inactivate p53 proteins
No DNA being repaired
Remember what NTP and dNTP stand for, and which bases are included in each
Nucleoside triphosphate:
ATP, GTP, CTP, GTP
Used for RNA
Deoxynucleoside triphosphate:
dATP, dGTP, dCTP, dGTP
Need to use for DNA synthesis
Distinguish the products and requirements of DNA versus RNA polymerases
DNA polymerase needs an RNA primer to extend the daughter strand from the extending polymer
RNA polymerase does NOT need a primer to begin
Both polymerases need monomers and a template to work
DNA polymerase needs dNTPs along with DNA strand in order for polymerase to link complementary nucleotides to the template strand
DNA polymerase makes DNA while RNA polymerase makes mRNA
Describe the relationships between DNA template, non-template, and coding strand
DNA template strand is what is being copied
DNA non-template strand is the opposite of the template strand
Coding strand is the same sequence and orientation as the non-template strand except it has uracil instead of thymines
Describe where codons and anticodons are found and their role
Codons are on the mRNA template, while anticodons are on the tRNA
Each triplet codon matches w/ a triplet anticodon on tRNA, which has the specific amino acid needed to make the protein we are looking for
Define cell cycle “checkpoints” (distinguishing checkpoints from Cdk/cyclin complexes), and give me some examples.
Checkpoints are the barriers that need to be overcome in order for the cell to go through the full cycle
Checkpoints monitor the progress of cell cycle events and prevent entry into subsequent stages until the current stage is completed
The Cdk/cyclin complexes are the molecules that trigger the next event by phosphorylation
G1 → S “restriction” point
Should a cell replicate its DNA?
This checkpoint is where a cell commits to divide OR will die
CAN NOT GO BACKWARDS (no other options) – can pause in G0 and go once they are ready
In multicellular organisms, there is an external signal (protein growth factor) from other cells that stimulates synthesis and expresses genes that encode cyclin and Cdk to drive transition for G1 → S
Cause the increase in cyclin concentration, which activates Cdk
Cdk and cyclin phosphorylate proteins responsible for S phase, leading to cell division
Signal amplification with receptor tyrosine kinases - cell proliferation
Ensures only get specific cells under appropriate conditions
M-phase checkpoint: spindle assembly
Pauses at metaphase in order to trigger anaphase
Without full chromosome attachment, stop signal is received
If mitosis fails, cancerous
If meiosis fails, aneuploidy - trisomies, monosomies (not normal ploidy)
Cohesin holds sister chromatids together at multiple points
Every chromatid attached to microtubules, then separase breaks cohesin down and microtubules pull so simultaneously breaks and pulls apart
Separase is activated by protein complex (ANAPHASE PROMOTING COMPLEX)
APC only active when all chromatids are attached to spindle
Chromosomes OR homologs do not separate until everything is proper
INDEPENDENT OF CDK OR CYCLIN
Mad and Bub proteins - regulate this checkpoint as they attach to chromatids not attached to spindle fibers
These proteins attract part of APC therefore, won’t be active
When APC is active, checkpoint is OFF
DNA damage checkpoint (look at objective below)
Explain why nucleic acid polymerization (synthesis) is always in the direction that it is
To build a polymer, you need an input of energy and monomers
The 5’ end is what has the phosphates which is what is the energy needed
Releases a phosphate and hydrolyzed
Therefore have to synthesize 5’ to 3’ because OH can’t be added to one phosphate because theres no energy input
DNA and RNA is always synthesized 5’ → 3’
Describe why an RNA polymerase is required during DNA replication AND Summarize the functions of helicase, single-strand binding proteins and topoisomerase
You need an RNA polymerase to make the RNA primer, which DNA polymerase needs to extend the DNA strand from the existing polymer AND
Helicase: unwinds the DNA duplex
Single-Strand Binding Proteins: stabilizes the single strands of DNA
Topoisomerase relieves the stress of unwinding
Name and describe the DNA sequences that begin and end transcription
Promoter is what starts transcription; there are sequences recognized by specific proteins for RNA polymerase to bind to and orient RNA polymerase
There's a TATA box (filled with thymines and adenines)
Transcription factors are a complex of proteins that bind to DNA at the promoter, then RNA polymerase bonds, creating transcription initiation complex, then RNA transcript starts to form
To end transcription, you have a terminator
Polyadenylation signal that proteins recognize and cut transcription a little bit past in order to add the tail
Explain the idea of reading frame AND Describe how a stop codon leads to terminating translation
Reading frame is the 3 nucleotides that encode a specific amino acid
If one nucleotide gets inserted or deleted, the frame is not the same anymore
If a nucleotide just gets substituted, the frame is still intact
AND
When the ribosome reaches a stop codon on the mRNA, the protein, or the release factor, is what recognizes the stop codon
It promotes hydrolysis and frees the newly synthesized polypeptide chain
Ribosomal subunits and other components all dissociate (can be used again!)
Tell me everything about p53.
p53 is a tumor suppressor that prevents cancer
Normally p53 in the nucleus is NOT phosphorylated and is rapidly exported from nucleus and degraded (continual process)
Make in cytoplasm, transport into the nucleus, target for destruction outside of the nucleus
DNA damage activates protein kinases that phosphorylate p53, blocking the degradation process
This makes p53 rapidly increase b/c you keep making it but you aren’t breaking any down
As p53 levels increase in the nucleus, p53 acts as a transcription factor to
1. Turn on genes that inhibit the cell cycle
2. Induce expression of DNA repair enzymes
3. If damage too extensive, can trigger apoptosis
Inhibiting the cell cycle gives the cell time to repair the damaged DNA
Describe the general structure of nucleosides and nucleotides, distinguishing those of DNA from those of RNA, remembering which are purines and which are pyrimidines
Nucleoside = nucleotide without the phosphate group (base linked to sugar)
Nucleotide: phosphate group bonded to 5’ carbon sugar which is binded to a nitrogenous base
RNA would have ribose while DNA has deoxyribose (look at 2’ carbon)
Uracil is only in RNA while thymine is only in DNA
Purines: adenine and guanine (two rings)
Pyrimidines: cytosine, uracil, thymine (one ring)
G-C have 3 H bonds ; A-T have 2 H bonds
Describe what “semi-conservative” replication of DNA means and its significance
Semi-conservaitve means DNA is replicated, and the two new DNA helices have one old strand (conserved) and one new strand therefore, semi-conservative
Both daughter helices are identical though; original molecule is copied in its entirety
Describe the features and functions of introns, and relate alternative splicing of mRNA to protein structure and function
Introns do not code actual protein products
they allow for alternative splicing, making it possible to generate multiple proteins from a single gene
Explain the role of tRNA molecules and aminoacyl tRNA synthetases
tRNA interprets the sequence and translates the mRNA into a polypeptide
Each tRNA molecule has an amino acid attached to it (meaning it is “charged”) - not electrically but charged meaning it has an amino acid or it doesn’t
Aminoacyl tRNA synthetases is responsible for the genetic code and connects the amino acid to its particular tRNA
Specific to each amino acid, therefore there are 20 different synthetases for 20 diff amino acids
Describe how cell fusion and microinjection experiments led to the identification of cytoplasmic factors regulating the cell cycle, and predict the outcome of such experiments (just tell me what you can!)
Cell fusion experiments: When two cells were artificially fused together and they were in different phases
Both nuclei rapidly went to the same phase of the cell cycle
Ex. cell in S phase fused with G1 → G1 nucleus immediately entered S phase and DNA was synthesized
Cell in M phase fused with G1 → G1 nucleus began mitosis without chromosome duplication (skipped over S phase)
Only went in the direction of the cycle - CAN’T GO BACKWARDS
Conclusion: molecules present in the CYTOPLASM control the progression to S and M phases
Microinjection experiments:
Cytoplasm from one cell into another (microinject and fractionate)
Biologists then purify proteins (could be through multiple methods like biggest → smallest) which help characterize and identify different factors in cells
Did this with frog egg cells
Found that there was a protein complex that triggered egg maturation in meiosis which was called the MPF (maturation promoting factor)
Remember the significance of the 1’, 2’, 3’ and 5’ positions of ribose and deoxyribose AND Recognize the 3’ → 5’ or 5’ → 3’ directionality of a nucleotide and nucleic acid
1’ → nitrogenous bases attached to this carbon
2’ → on the ribose, has an OH group; on the deoxyribose, has an H group
3’ → has an OH group that links to the next nucleotide
5’ → has the phosphate group (outside of ring)
AND
3’ end has the OH group attached to it
5’ has phosphate groups
Once added to a DNA molecule, phosphate group (1) at the top is attached to the “bottom nucleotide” at the 5’ end then 3’ attached to the new nucleotide
Explain when, where and why telomerase activity is required, and draw its activity
The leading strand replicates the whole template strand, but the last RNA primer on the lagging strand sits near the end of the template strand
When RNA primer is removed, a section of DNA is unreplicated, therefore if you kept going, chromosomes would keep getting shorter and shorter
Telomerase is an enzyme that contains an RNA template that allows the template strand to be lengthened by telomeres
A new segment of the lagging strand is formed so that all of the DNA can be entirely replicated
Don’t have access to the telomeres as they get folded
Compare and contrast DNA replication with transcription regarding purpose, location, requirements (enzymes & nucleotides), directionality, and templates
Purpose: similar to DNA rep where we are synthesizing a nucleic acid but DNA rep is making DNA while transcription makes RNA
Location: both are in the nucleus!
Requirements: transcription only needs RNA polymerase and NTPs while DNA replication needs DNA polymerase AND RNA polymerase (to make primers) along with dNTPs
Directionality: both get read 3’ → 5’ while both get synthesized 5’ → 3’
Templates: for DNA replication, both strands are templates along the entire chromosome; for transcription, there is only one strand of DNA that is looking at specific genes (not the entire chromosome)
Explain and distinguish the two mechanisms resulting in genetic code redundancy
“Wobble” → the same tRNA with the same amino acid can recognize different codons through this
Diff codons are making the same amino acid as the first two are the same and the last one can just dangle
Several different tRNAs get linked to the same amino acid but recognize different amino acids (has the same aminoacyl-tRNA synthetases with the same amino acid)
DIFFERENT CODONS SPECIFY THE SAME AMINO ACID - redundancy without ambiguity
Describe the activity of MPF and its cyclin/Cdk components in driving cell cycle transitions
MPF was the maturation promoting factor
Made of cyclin and Cdk (cyclin-dependent kinase (enzyme)) - this is when its active
Only one type of Cdk complex
Specifically triggers the cell cycle to go from G2 to M phase (mitosis and meiosis)
Driven by kinase activity (phosphorylates things for them to activate / deactivate)
The kinase activity drives cells into meiosis and also has another protein involved where concentration in the cell varies with cell cycle (called cyclin!)
When cyclin concentration is high, so is MPF activity
Cyclin binds to Cdk, forming MPF, triggering cell cycle to go into meiosis
Cyclin regulates Cdk
MPF targets / effects (through phosphorylation):
“Condensins”
Once they are phosphorylated, they are active
Initiate prophase, therefore they condense chromosomes
Nuclear lamins
For prometaphase:
Helps break down nuclear envelope by depolymerization after the lamins get phosphorylated
Centriole proteins
Anaphase promoting complex (APC)
Transition from metaphase to anaphase
After the checkpoint, there is a targeted destruction of protein (in this case, cyclin) for things to rapidly be undone
When cyclin is being degraded, the concentration drops (regulation by destroying cyclin and keeps the cycle going forward → then everything goes back to “normal”
Describe the major discoveries of Chargaff, Franklin, and Watson & Crick
Chargaff:
if you add up the percentage of purines, it should equal 50%
If you add up the percentage of pyrimidines, it should equal 50%
Amount of adenine is similar to thymine; vice versa with cytosine and guanine
Add all 4 up = 100%
Franklin
Started the research to determine that DNA was a double helix (x-ray crystallography)
Also started suggesting about the width of the helix
She concluded that sugar-phosphate backbones are on outside of DNA molecule
Watson & Crick
The backbones are antiparallel to each other
Then they declared that it was a helix
Purine has to be paired with a pyrimidine (therefore A and T with C and G)
Also has to be that way because of amount of hydrogen bonds each can make
Illustrate Meselson and Stahl’s predictions based on three hypotheses of DNA replication including their actual results and evidence for semi-conservative replication
Hypothesis 1: semiconservative replication; Hypothesis 2: conservative replication (1 fully original strand and 1 new); Hypothesis 3: dispersive replication (mix of new and old DNA)
First grew cells in medium with 15N and collected and purified sample
Then transferred it to 14N, collected and purified sample
Then after dividing a second time in 14N, collected and purified sample
Centrifuge and compare locations of DNA bands
If semiconservative, we would have ½ low density (all 14 N) with ½ intermediate-density DNA
If conservative, ¼ high density (15N) and ¾ low density
If hybrid, all would be intermediate density DNA
Results: after two generations, ½ low density and ½ intermediate density DNA
Replication is semiconservative
Describe the major features and purposes of pre-mRNA processing in eukaryotes
Pre-mRNA processing is done in the nucleus before the mRNA can leave to go to the cytoplasm and perform translation
Part of export out of the nucleus and stabilizes the mRNA (won’t break down as easily), which enhances translation
In eukaryotes
Guanine cap is added to the 5’ end
poly-A tail added to the 3’ end (gets added on after transcript is cut - not encoded from DNA we just saw)
Introns and exons! Introns are cut out and removed from primary transcript, exons are spliced together
Describe and distinguish silent, missense, nonsense, and frameshift mutations
Substitution
Silent → no amino acid change
Missense → diff amino acid
Nonsense → specifies stop (early finish to the protein - non functional?)
Insertion or Deletion
Frameshift → changes reading frame
If it changes every single amino acid after, massive missense
If it changes the next amino acid to stop, massive nonsense
If 3-nucleotides get inserted or deleted, you are missing an amino acid but not a frameshift