Random shit to memorize week 2 Flashcards
Start codon
AUG, encodes methionine
Stop Codons
UAA, UAG, UGA
IF1 and IF3 bind to _________
30S subunit of ribosome
IF2
delivers formyl-methionine tRNA to P site to pair with AUG codon
E1
binds aminoacyl tRNA and brings it to the A site
Peptidyl transferase center
catalyzes peptide bond formation using energy from charged tRNA
E2
binds and transfers tRNA from A site to P site
What kind of energy and how much is used to add each AA in translation?
GTP and ATP
4 per AA
(2 ATP to charge tRNA, 1 GTP to deliver tRNA to A site, 1 GTP to translocate A–>P site)
Prokaryotic Translation
Shine-delgarno
Polycistronic
Eukaryotic translation
- Cap-dependent, IF4E protein
- Kozak sequence
IF4E protein binds _________ and brings in ________
binds 5’cap on mRNA, and brings in IFs for translation
Interferon
released by cells under viral attack, and signals to nearby cells to prepare for infection by virus
Interferon causes nearby cells to activate ___________ and phosphorylate _________ prevent __________
1) activates viral mRNA endonucleases
2) phosphorylate eIF-2, prevent delivery of tRNA-Met to start site
apoB
mRNA edited post-transcriptionally to alter function - mRNA in intestine processed by enzymes so it is shorter than mRNA in liver
Rapamycin is a cancer treatment that prevents __________
phosphorylation of 4E-BP, preventing formation of translation initiation complex on the 5’cap
Transferrin
When Iron is low, transferrin level ________
When Iron is high, transferrin level ______
transports iron into cell
Low iron = increase transferrin
High iron = decrease transferrin
Ferritin
When Iron is low, Ferritin level ________
When Iron is high, Ferritin level ______
Sequesters iron
Low iron = decrease Ferritin
High iron = increase Ferritin
Iron response binding protein binds __________ at low iron levels, stopping _______ and stabilizing ________
Iron Response Element
stopping ferritin production
stabilizing transferrin
When iron levels are high, iron binds the ________ and allows ________ to be translated and _________ to be degraded
Iron response binding protein
allows ferritin to be translated
transferrin degraded
Both insulin and keratin us ________ as an integral part of their structure
disulfide bonds
Hydroxyproline
- Stabilizes structure of collagen via H-bonds
- Production is Vitamin C dependent, so no Vitamin C = scurvy
Gamma-Carboxyglutamate
Present in blood clotting proteins, Vitamin K dependent, so without vitamin K = bleeding disorders
Coumadin
Targets enzyme that modifies AA into gamma-carboxyglutamate
Glycosylation adds a sugar onto the O of ______ and _____ and the N of ________
Ser, Thr
Asparagine
Phosphorlation occurs on _______, _______, and _____ and is important in signal transduction
Ser, Thr, Tyr
Gleevec
inhibits kinase activity of bcr-abl and stops continuous production of WBC – treats chronic myelogenous leukemia
Ubiquination
ubiquination of protein marks protein for degradation by proteasome
Bortezomib
proteasome inhibitor, prevents cancer cells from replicating
Sickle Cell Anemia is caused by __________ which makes hemoglobin molecules aggregate
single AA change in hemoglobin
Angiotensinagen protein precursor is cleaved by ________ into _________, which is then cleaved _______ to make __________
Renin protease –> Angiotensin I
ACE –> Angiotensin II
Collagen has a unique ________ rich in _____ and _______ and is dependent on ___________
triple helix structure, rich in Gly and Pro and is dependent on vitamin C for hydroxyproline production
Kd
dissociation constant = [ligand] when 50% of ligands bound
Bohr Effect
O2 binds better to hemoglobin at higher pH, and is released well at lower pH
Two classes of chaperones
Hsp70/Hsp40 and Chaperonin
Hsp70/Hsp40 chaperone helps protein folding by….
Use ATP?
binding to hydrophobic regions of unfolded protein to prevent aggregation
Uses 1 ATP
Chaperonin helps protein folding by a several step process:
Uses ATP?
protein binds to uncapped site → ATP used to move cap over it → protein folds → ATP used to remove cap and release protein
Uses 2 ATP
Protein disulfide isomerase
Breaks improperly folded disulfide bonds, allows correct reformation
Prolyl Isomerase
Speeds up proline conversion between cis and trans conformations
Gel filtration chromatography
protein purification based on size
Beads with small pores, takes small molecules longer to run through column
Ion Exchange Chromatography
protein purification based on charge
Cation exchange column → - charge beads bind + target protein
Use salt to disrupt binding and elute
Affinity Chromatography
protein purification based on ligand binding
- Protein binds resin with ligand attached
- Elute with solution with high concentration of ligand
Gel Electrophoresis
protein purity analysis
- SDS polyacyrlamide gel used to separate proteins by size
- Charge applied to gel, and large proteins move slower
Edman Degredation
Determines sequence of AA from N terminus
miRNA functions in ____________ and ________
translational repression, mRNA degredation
miRNA originates from __________ and uses ______, _______, and ________ proteins
pre-miRNA precursor molecule
Drosha, Dicer, Argonaute
siRNA functions in _______ and __________ and requires ________
mRNA degredation, transcriptional silencing
requires a perfect match with target
siRNA originates from _______ and uses ______ and ______ proteins
dsRNA
dicer, argonaute
when miRNA matches perfectly with the RNA it __________ and when miRNA matches imperfectly with RNA it __________
Imperfect match = RNA degradation OR translational repression
Perfect match = RNA degradation
piRNA function in _________
transcriptional repression
piRNA originate from ________ and are _______ and _______ independent
ssRNA
Dicer and Argonaute independent
Long non-coding RNAs are > __________ NTs long
200
Argonaut/RISC
RNA induced silencing complex
-Cleaves/degrades RNA
Dicer
ribonulcease that cleaves miRNA precursor and dsRNA molecules
Drosha
Rnase enzyme, processes newly transcribed primary miRNA in nucleus
Restriction Fragment Polymorphism is used to diagnose certain diseases like __________ by __________
- sickle cell anemia (mutation –> longer DNA strand)
- by differentiating DNA sequences of different sizes
Southern Blot
looking for specific DNA sequence, use cDNAp probes
Northern Blot
looking for specific RNA, use anti-sense cDNA probe
Western Blot
looking for specific protein, use antibody probe on SDS-PAGE gel
3 stages of PCR
1) Heat to 95 degrees C (denaturing DNA).
2) Cool to 55 degrees C (allows primers to hybridize).
3) Warm to 72 C at which time the polymerase (Taq polymerase) copies the DNA from dNTPs in solution
PCR vs. Sanger Sequencing (similarities/differences)
- Similarity: both use primers to initation replication
- Difference: PCR uses dsDNA, sequencing uses ssDNA and ddNTPs
Plasmids
- vectors, amplify DNA sequences in bacteria
- Max insertion of 20kb into E. coli
- Transformation is inefficient
Bacteriophage
infects E. coli, in order to use its replication machinery to produce recombinant vector (much more efficient transformation)
Cosmids
hybrid of bacteriophage and plasmid: use plasmid replication origin; can take up to 45 kb insert into E. coli.
BAC vector
bacterial artificial chromosome with insert up to 300kb into E. coli, good for chromosome mapping and sequencing
YAC vector
yeast artificial chromosome with insert up to 2mb, chromosome mapping and sequencing
Retroviral Vector
can carry very large inserts; introduce DNA into mammalian cells, delivers gene therapy.
Microarrays are used to measure _______ but tell you nothing about _________
mRNA levels (gene expression) nothing about SIZE of mRNA
Retinal Blastoma Protein
inhibits cell cycle transition from G1 to S
-Loss of RB = tumors
CDK4 + cyclin complex inhibits ________ and allows _________
inhibits RB, and allows cell cycle to advance into S phase
Assembly of Pre-RC at the origin of replication occurs only in _______ when _________
G1
CDK is inactivated
Origin recognition complex
binds origin of replication, landing pad
MCM Helicase
recruited in G1 to pre-RC complex, activated when cell enters S phase and CDK is activated
Activation of Pre-RC occurs only in ________ when _________
S phase
when CDK is activated, and MCM helicase is phosphorylated
p53 activates _______ which prevent _________. If mutated then…
CDK inhibitors
prevent entry into S phase
If mutated = uncontrolled replication, cancer
ATM/ATR are protein kinases that phosphorylate ______ , causing _______
p53
p53 activation, prevents entry into S phase
Short read sequencers
- Produce millions of short 100 base-pair reads
- Use 1000 bp DNA templates
- Low error rate
- best for analyzing genotype
Long read sequencers
- Produce 10,000 sequencing reads, up to 10,000 bp in length
- High error rate
NHEJ
- Ends protected
- Occurs anytime in cell cycle
- Imperfect
- Regulated by 53BPI
- Uses: Ku, Artemis complex, Polymerase, Lig-4
Ku is used in ________ and acts to __________
NHEJ
acts to recruit DNA PKcs (protein kinase)
Artemis complex
processes ends in NHEJ, makes ends suitable for ligation
Lig-4
ligase used to restore phosphodiester bonds in NHEJ
HR
- Perfect repair
- Ends resected
- Requires presence of sister chromatid
- Only occurs in S and G2 of cell cycle
- Leads to genetic diversity in Meiosis
- Regulated by BRCA1
Misregulated HR can lead to __________ if __________ are used instead of _____________
loss of heterozygosity
homologous chromosomes
sister chromatid
Normal pathway of Amyloid Precursor protein
APP cleaved by alpha-secretase and then by gamma-secretase
Abnormal pathway for Amyloid Precursor protein
APP cleaved by beta-secretase and then by gamma-secretase → AB40 or AB42 (much more toxic)
Apolioprotein E
- enzyme that processes/clears out B-amyloid
- 3 allele variations, and one of them (apo 4) is not very good at clearance
Cofactor
metal ions bound within the enzyme, used by enzyme to do some chemistry
Coenzyme
small molecule bound in enzyme that may provide a functional group to participate in the chemistry
Km = ?
High Km =
Low Km =
Km = [S] at ½ Vmax
- High Km = enzyme has low affinity for substrate
- Low Km = enzyme has high affinity for substrate (requires less substrate to achieve
Kcat = ?
turnover number, # substrate converted to product in a given time when [S] is at saturation
Kcat/Km =?
efficiency of an enzyme
-Higher number = better enzyme
Competitive Inhibitor
binds only to enzyme, competes with substrate for the active site
-Increases Km
Uncompetitive Inhibitor
binds ES complex, does not bind active site (binds elsewhere)
-Lowers Vmax, changes Km
Mixed Inhibitor
binds outside the active site, but can bind either E or ES
-Effects both Km and Vmax
Irreversible Inhibitor
act to permanently change the enzyme into a non-functional form
-EX) penicillin
4 Mechanisms for Enzyme Regulation
1) Allosteric regulation
2) Covalent modification
3) Binding of another protein
4) Proteolytic cleavage
BRCA1 regulates ______, and recruits endonucleases and exonucleases when __________
Homologous Recombination
Phosphorylated by ATM
Mutation in BRCA1 and 2
cell checkpoint is defective, DNA damage not repaired properly –> chromosome breakage and abnormal recombination
-Without BRCA1, 53BPI pushes repair towards NHEJ (more errors)
IRES
involved in cap-independent translation initiation
Non-polar AA (6)
Glycine Alanine Valine Leucine Methionine Isoleucin
Aromatic AA (3)
Tyrosine
Tryptophan
Phenylalanine
Polar AA (6)
Threonine Serine Cysteine Proline Asparagine Glutamine
Acidic AA (2)
(-) charge
Aspartate
Glutamate
Basic AA (3)
(+) charge
Histidine
Arginine
Lysine
