Cell E3 (1)

How does the DNA double helix and base pair rules lead to a mechanism of DNA replication?

we know that the amount of A=T and C=G,, this can show that one strand could be used as a template for another strand

What is the origin of replication? Why is the consensus sequence for an origin of replication AT rich?

AT bonds are less strong than GC bonds, where you start DNA replication you need to separate strands so AT makes that easier, this is the place replication starts (signals for DNA polymerase)

What does it mean that DNA replication is bidirectional?

polymerase will replicate both strands of DNA to make 2 new strands, and will always add 5’→3’ on both, but since the strands are antiparallel, polymerase will move two different directions

Which direction does DNA polymerase replicate DNA and why?

5’→3’,, it needs an OH group to add another nucleotide to

Difference between leading and lagging strand, how are they replicated?

leading allows for only one primer to synthesize DNA 5’ to 3’, a countinuous replication while lagging needs multiple primers and replicates toward replication fork, both need primers to be taken out and DNA polymerase to fill in

What is a telomere and why are they necessary?

the end part of linear DNA, usually heterochromatin, they protect the ends of the chromosome from being lost

What is telomerase/what does it do?

catalyzes additional copies of the telomeric repeat sequence, which compensates for gradual shortening in replication

In what cells is telomerase active, what happens to cells if there is no active telomerase? How does this explain cellular senescence?

mainly in germ cells/stem cells that divide many times, if there is no telomerase, the cells could start loosing some genetic material (senescence)

How can DNA be damaged? What are natural processes that lead to DNA damage and how do they damage DNA, how do mutagens damage DNA?

spontaneous (transient formation of tautomers, slippage during replication, spontaneous damage to individual bases) or mutagens (mobile genetic elements, chemical/radiation), tautaumers lead to shift, depurination (A or G lost), ionizing radiation (removes electrons)

base excision repair

corrects single damaged/wrong base, very accurate, removes wrong base, repair polymerase comes in and is filled in

nucleotide excision repair

remove a few bases, very precise, uses an endonuclease to remove some bases, and then polymerase fills it in again

mismatch repair

when bases are mismatched, removes the single nucleotide and fills it in with right one, precise, can know what is the old vs new strand

translesion synthesis

error-prone, when there is a lesion (from UV or other damage), when DNA polymerase can’t recognize the bases because of this, a different polymerase will come in and move other one out of the way and repairs as best as it can

nonhomologous end-joining

error-prone, repairs double strand break, proteins will bind the ends and trims them (loss of nucleotides) and then forms them back together

synthesis-dependent strand annealing

if you have a replicated chromosome and get double strand break on one of the two, one of the broken strands can go to sister chromatid and be copied, usually not error-prone (sister chromatid should be the same)

homologous recombination

repairs double stranded break by using another chromosome (diploid so if mom chromosome broke, goes to dads), as template, crossing over can occur, in theory shouldn’t be too error-prone but if the info is different it will be “error prone”

How does CRISPR work to induce mutations in DNA in animal cells?

Cas9 is a protein that will contain a guideRNA (which can be designed) to bind to a certain part of DNA and then create a double stranded break, can use non homologous end joining (creating mutation), or uses homologous recombination (and you give it DNA), so new DNA is made in place of original

How can problems in DNA repair lead to human disease, including cancer?

if you can’t do base excision repair it results in fetal death, when cell can’t repair errors, there is an increased rate of mutations which disregulates cell cycle and increases risk for cancer

What are dNTPs?

a nucleotide but with three phosphates

Where does translation/transcription occur in prokaryotes vs eukaryotes?

Where is the N and C terminus of a protein?

N will be at beginning, at amino group of protein, c terminus will be at the end

What are the five levels of regulation of gene expression?

genome, transcription, RNA processing, translation, post-translation

How can chromatin structure regulate gene expression?

via histone modification, can change how something is expressed

What effect do histone acetylation/deacetylation, histone acetyltransferase/deacetylase activity, and DNA methylation have on gene expression?

acetylation will add an acetyl group which will make DNA easier to access/transcription easier to occur, while DNA methylation is adding a methyl group and often it will condense the DNA or repress it

What is epigenetics and what is the underlying mechanism?

stable alterations in gene expression that are transmitted from one cellular generation to the next without requiring changes in a gene’s underlying base sequence, such as methylation which is inherited

What is differential gene transcription and why is it important?

the transcription of different genes to produce different sets of RNA, demonstrates how different types of cells produce different proteins

What is a promoter and what are proximal control elements?

promoter - a region of DNA near the transcriptional start site, transcription factors assemble with RNA polymerase here, pce are where transcription factors can bind

How do regulatory transcription factors regulate gene expression? Roels of enhancers and silencers?

gene sequences (transcription factors) that bind to proximal control elements which increase or decrease transcriptional initiation by interacting with components of the transcription apparatus,, enhancer stimulates gene transcription and silencers decrease it

In what ways can gene expression be regulated post-transcriptionally?

RNA processing

What is RNA processing?

what happens to RNA before it is ready to be sent for translation, addition of a 5’ cap, a 3’ poly a tail, and splicing which takes out introns and leaves exons

What is alternative splicing and how can it regulate gene expression?

the cells ability to permit some splice sites to be skipped and others to be activated, different sets of exons left in

What are various translational control mechanisms?

RNA interference, microRNAs, these regulate the rate at which mRNA is translated into protein, alters ribosome or protein synthesis factors, regulate activity, stability of mRNA

What is RNA interference and how does it regulate gene expression? (roles of Dicer, RISC, and argonaute proteins)

dicer cleaves ds RNA into an siRNA (short), the siRNA is combined with RISC proteins (contain argonaute), one of the two RNA strands then degraded, either siRISC binds to mRNA or siRISC enters nucleus and binds to DNA inhibiting transcription, if binds to mRNA exactly degredation of mRNA if binds to mRNA partially inhibition of mRNA

What are microRNAs and how are they involved in regulation of gene expression?

single stranded RNA’s that can bind to/regulate expression of mRNA produced by genes that are separate from the genes that produce the mRNAs

How is protein degredation regulated? What is the role of ubiquitin, ubiquitin ligase, and proteasome?

its activated by attaching ubiquitin to a ubiquitous activating enzyme in an ATP dependent reaction, the activated ub is transferred to a ubi conjugating enzyme and linked (ligase) to lysine in target protein, additional molecules of ubiquitin are added making polyubi chains which serve as a targeting signal that are recognized by proteosomes

What is differentiation/differentiated cells?

distinguished from each other based on differences in appearance and the products they synthesize

What is Drosha?

cleaves the primary miRNAs into smaller hairpin RNAs, then these are exported into the cytoplasm where dicer cleaves the hairpin loop to produce 2 complimentary RNA molecules

What are second messengers and how are they used?

they relay signals from ligand binding to the inside of cell which initiates changes

examples of second messengers

cyclic AMP and IP3

What is receptor affinity? What are high and low affinity receptors?

this is the relationship between concentration of ligarnd in solution and number of receptors occupied, high affinity when almost all of the receptors are occupied at low concentrations of free ligand, low afinity when takes a high concentration of ligand for most receptors to be occupied

How can receptor signaling be terminated, how can they be turned off?

by removing or inactivating the ligand/things that drive the pathway, or turning GTP to GDP

How do signal transduction pathways often lead to signal amplification?

signal amplification is the overall process by which a cell converts a signal into a functional response in the cell, amplification happens where the number of activated molecules or second messengers increases at each step

What are G protein coupled receptors and how do they work? What is the signaling pathway they activate?

usually transmembrane domains, coupled with w/ Ga, Gb, Gg, heterotrimeric G proteins

What are G proteins and how do they work?

act as switches to relay signals, proteins that are either on or off depending on if GTP or GDP is bound to it

What are the roles of G alpha, G beta, and G gamma?

the three basic subunits of a G protein, alpha is what binds to GTP or GDP, when it is active and bound to GTP, the other two detach from it and the g protein can continue with the signal

Gs pathways, What downstream signaling do they activate?

Gs binds gtp and dissociates from g beta and g gamma, GTP-Gs/a complex binds to and activates adenylyl cyclase which synthesizes cAMP, which can bind to protein kinase A and can phosphorylate some substrate

Gq pathway, what downstream signaling does this activate

when gtp binds, beta and gamma dissociate, the gtp/a complex binds to phospholipase C, activating it and causing cleavage of PIP2 into IP3 and DAG, IP3 released into cytosol triggering calcium release, DAG remains in membrane where it activates protein kinase C

Gi pathway, what downstream signaling does this activate

when gi (inhibits) is bound to gtp, it inhibits adenylyl cyclase

What reaction does adenylyl cyclase catalyze? Why is this important?

the conversion of atp to cyclic amp and ppi, this is important for turning glycogen to glucose in response to epinephrine (used to activate protein kinase A)

What is a kinase and what does it do?

they transfer phosphate groups to a protein (usually from atp or high energy molecule)

What are the two major classes of kinases and what amino acids do they act on?

tyrosine kinases (phosphorylate a tyrosine residue) and serine-threonine kinases (phosphorylate serine or threonine)

What are receptor tyrosine kinases? What is the signaling pathway they activate?

What is the role of autophosphorylation in receptor activation?

this is the process of receptors phosphorylating other receptors of the same type (in receptor tyrosine kinases)

What does it mean that signaling pathways are integrated?

different signaling transductions/cascades interact, components from one can activate/inhibit/affect another (crosstalk)

What is the effect of hormone epinephrine? What are the cellular effects?

released when in high stress situations, prepares body for dangerous or stressful situations, it stimulates the breakdown of glycogen and supplies glucose to the muscles

What is the signaling pathway activated by epinephrine? How is signal amplification evident in this pathway?

Gs protein receptor accept the receptor is a B-adrenergic molecule, activates Gs, activates adenylyl cyclase which makes cyclic amp and that binds to protein kinase A, which phosphorylates phosphorylase kinase making it into active phosphorylase, which can convert glycogen to glucose, protein kinase A also inactivates/phosphorylates glycogen synthase

What is the role of insulin? What are the cellular effects?

What is the cell signaling pathway activated by insulin?

receptor desensitization

cells can change the density of receptors in response to a signal, removal of receptors can happen via receptor mediated endocytosis, process where a cell’s response to a signal increases even if the signal is there still

agonist vs antagonist

agonist (activate receptor) antagonist (inhibit receptor)

receptor mediated endocytosis

this can be a way for receptors to be removed from cells, when small portions of the plasma membrane containing receptors inviginate and are internalized

signal cascade

cAMP

made by adenylyl cyclase and activates protein kinase A

protein kinase A

activated by cAMP, it phosphorylates phosphorylase kinase and also inactivates glycogen synthase in epinephrine pathway

phosphodiesterase

this inactivates/cleaves cAMP when epinephrine pathway needs to end

phospholipase C

insoitol triphosphate (IP3)

calmodulin

a calcium sensitive enzyme, two calcium ions bind to it and it undergoes a change in shape that forms the active complex, when a protein is present that contains a calmodulin binding site, calmodulin wraps around it influencing the function of the protein

Ras

MAP kinase

B-adrenergic receptor

the receptor is g-protein coupled and accepts epinephrine as ligand

glycogen phosphorylase

phosphorylates glycogen and makes glucose-1-phosphate which is usable form

glycogen synthase

creates glycogen, is inactivated by protein kinase A during epinephrine pathway in order to make glucose rather than glycogen

akt

PTEN