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107 Cards in this Set
- Front
- Back
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What is the function of different RNA's involved in translation?
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tRNA=important for transferring amino acids to the ribosome to make a poly peptide.
rRNA-makes up different subunits of the ribosome 31 for different amino acids |
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Explain the structure of a tRNA
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acceptor arm at the 3' end
anticodon is 3' to 5' Does not employ normal 4 bases |
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How and why is wobble achieved?
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Allows for mutations w/o change of function of the protein
Allows for non standard base pairing, U with A or G, G with C or U or I with UC OR A Based on alternative hydrogen bonding |
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What enzyme charges a tRNA?
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Amino acyl synthetase
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Explain pre initiation of translation
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the ribosome must find the Shine Delgaro (prok) or the Met cap and go to AUG (euk)
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Explain translation initiation:
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small subunit lines up with the AUG start tRNA P come in and binds to the AUG, large subunit attaches tRNA w/ a specificed aa enters the A site
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Elongation:translation
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Peptidyl transferase attaches the amino acid in the P site to the A site.
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What enzyme forms the peptide bond?
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Peptidyl transferase.
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How does termination of translation occur?
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The release factor binds one of the stop codons UAA UGA UAG and causes release of ribosome and therefore ends translation.
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What essential molecule is necessary for translocation?
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EF2 (Elongation factors can be inhibited)
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What are subunits in prokaryotic DNA vs Eukaryotic DNA?
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Prokaryotic ribosome has a 30s and a 50s (70 total) and can perform polycistronically
Eukaryotic ribosome has 40 and 60s (total 80s) and performs monocistronically |
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What does the P site on the ribosome do?
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Contains the growing peptide chain
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What does the A site on the ribosome do?
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It is the donor tRNA amino acid.
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What is the E site in the ribosome?
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Exit site.
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Explain how ubiquination works?
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Ubiquitine is activated by e1, transferred to 32, transferred to lysine by e2 U ligase
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What is the purpose for ubiquination?
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targets the protein for degredation by the proteosome
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Explain the structure of the proteosome
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19s cap U binding and ATP acitvity, 20s proteases,
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Tetracyline
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affects the A site, prevents tRNA binding
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streptomycin
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bacterial 30s subunit prevents initiations
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erythromycin
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50s subunit, blocks translocation
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chloramphenicol
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messes up bacterial peptidyl transferase
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Puromycin
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Premature termination
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Cyclohexamide
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Eukaryotic 80s irbosome peptidy transferase
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Why do antibiotics sometimes mess people up?
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Our mitochondria are basically prokaryotes.
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Diptheria toxin
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ends trasnlocations
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How are proteins directed to specific areas of the cell?
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They have sequences that direct where they end up.
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Alpha helix
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Dipole, bottom is negative top is positive, each carbonyl acts on the NH 4 residues down, antiparallel satisfies electrostatic attraction. 3.6 AA per turn PROLINE WILL BREAK
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Beta Sheet
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Macro dipole because c=o point down, an be parallel or antiparalell
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B turn
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most common type of non repetitive structure 25% of residues in a protein are in a b turn
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Phi
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alpha c to N
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Psi
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alpha c to c=o
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Ramachandran plot
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shows islands of stability for phi and psi angles, dark blue areas are the possible motifs, (upper right is the left alpha helix) upper left is the right twisted sheet etc.
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SAA
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Serum amyloid peptide, has a right handed twist but is is supposed to be left, due to a change in chirality, causes alzheimers
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How do proteins relate to cancer?
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50% of cancers occur
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PDZ domains
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recognize the C terminal sequence
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Ankyrin
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repeats evolved fit for many proteins
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Immunoglobulin domains
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evolved fit for many proteins
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DNA binding domains
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eg zing finger
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Kinase Domains
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Variable phosphorylation
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Erythrocyte membrane skeleton (spectrin)
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Skeleton of filamentous proteins that cross link short actin filaments that underlie the plasma membrane. 106 repeats.
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Dimeric spectrin
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is formed by the lateral association of alpha nad beta monomers to form a dimer,
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Tetrameric spectrin
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Forms when dimers associate in head to head formation, creates hexagonal complexes
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Primary, Secondary, Tertiary, Quaternary
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-sequence, 2 alpha helix, 3, repeating of alpha helix, 4 dimers + tetramer
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HS
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messed up band 3 protein so no ankyrin binding to spectrin therefore the lipid bilayer not anchored (verticle) issue NO WHITE DOT IN THE MIDDLE
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HE
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cylindrical RBC, horizontal issue where it cannot form the heterodimer, mutations on the C end of beta and the N end of alpha spectrin, perturbs the deformability (can become HPP)
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How do molecular chaperones function?
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They can act in variety of ways, they can prevent folding until translation is complete, catalyze, and protect from environment.
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How do HSPs work?
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help fold by altering the polarity of the interior surface of the HSP, assist proper folding in 10% of proteins
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Group 1 chaperonins
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prokaryotic, GROEL has a LID called GROES (needs more energy that the euk version)
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Group 2 chaperonins
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eukaryotics no detachable lid, apeture eg. TRIC
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GROES
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DETACHABLE LID
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Two types of polysomes
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Cytoplasmic-makes proteins for cytoplasm
ER-targets porteins to subcellular compartments via sequence |
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Signal Recognition Particle
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binds sequence on mRNA then is recognized by SRP receptor on ER and inserted into the membrane where translation will occur (allows proper folding)
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Hemocrit
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the % of volume of blood composed of erythrocytes
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Myoglobin
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binds O2 more tightly and does not display cooperative binding
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Hemoglobin
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Displays coop binding because one of the helices is affected by binding of the first oxygen changes comformation of the the other binding sites
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What happens to porphyrin site when o2 binds
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O2 binds distal histadine (attracts in) pulilng the F8 helix in that breaks salt bridges between heterodimers
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Negative Alloesteric effectors for hb
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H, 2,3 bpg, CO2, temperature increase
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Anfinsen experiment-
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shows that even if you get rid of teriary structure etc, denature, you can refold the same structure based on the primary sequence (shows that 1 sequence is important)
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2,3 BPG
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tri anion binds between subunits (can't really bind fetal hb) polyanion binds to his and lys side chains that stabilize the T format, (in fetal they lack acidic residues and therefore don't have the cations to bind this anion)
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CO2-
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carbamate favros salt bridge formation
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Proton H (lower pH)
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bohr effect, protons bind to the side chaisn and shift the quilibria to the T form
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Temperature
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increased temp shifts to the right, people are now flushed (not blue)
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What is the difference between cooperativity and Allosterism
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Cooperativity = homotropic effector makes curve sigmoidal, allosteric, heterotropic long distance binding makes curve shift left/right
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How is insulin activated by cleavage?
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Stars out as a single molecule (proinsulin) with a useless part in between alpha and beta subunit, S-S bonds form between a and b, and useless part is cleaved out.
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Example of reversible post translational modification
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phosphorylation
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Example of irreversible post translational modification
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ubiquination
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Examples of post translational modifications
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Assembly of proteins into higher order structure (blood clotting)
Activation of molecular activity (proteolytic etc) Subcellular targeting (prenylation) Molecular recognition glycosylation Reversible cell signalling (po4) |
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How many proteins are phosphorylated, how many acetylated
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Phos-10-50, acetylation 80-90
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Why is acetylation important?
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Gets drugs across the blood brain barrier
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2 ways to add hydrophobic groups to a protein
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myristylation-targets protein to the lipid bilayer
prenylation-farnesyl group, RAS in the plasma membrane (inhibit prenylation to inhibit cancer) |
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Glycosylation
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involved in cell to cell recognition- rh factor
can be useful for folding, keeps proteases away N-linked-bound to asparagine through amide linkages Signal sequence allows the protein to be translated directly into a membrane RER so that the folding can occur correctly, gets a MANNOSE, travel to the golgi |
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Glycation
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DIABETES-sugar reduces proteins in a haphazard fashion and impairs its functions (lens of the eyes, brain)
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What enzyme performs acetylation?
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Done by N Alpha acetylatransferase that takes it from acetyl coA
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Kinase structure
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ATP binding lobe (n terminus)
substrate binding lobe (large C terminal lobe) catalytic center activation loop (phos or dephos to activate) |
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How does the kinase work
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phosphate from at binding loop to the substrate
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What is the significance of a mutation in the atp binding part (ploop) of the kinase?
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Worse prognosis in CML because gleevec can not competitively inhibit that site. (atp agonist)
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PTP
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protein tyrosine phosphatases, have a CYs that forms a cleavable enzyme phosphate complex which is hydrolyzed to reform the enzyme and phosphate
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PP
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protein phosphatases-contain a dinuclear metal ion which may activate a water molecule that hydrolyzes
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DSPTSP-dual whatever
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they have big enough active sites to accommodate all the different side chains
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What is the role of kinase control in biological processes such as cancer?
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HER2 receptors are tyrosine kinase receptors implicated in cancer, they are overproduces and therefore more likely to idmerize and cause uncontrolled growoth
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What is gleevec (imatinib)
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competitive inhibitor of tyrosine kinase hers (agonist of atp) -not as effective if there is a mutation in the p-loop atp binding site.
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2 cancer pathways
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proliferation, resist apoptosis
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Michaelis Mentin equation
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Vo= (vmax[s])/(km + [s])
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Assumptions of MM equation
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E,S, ES are in equilibrium,
S > E S binds to 1 site only Conversion to E + P is rate limiting E + ES=Etotal |
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Lineweaver burke
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X intercept = -1/KM
Y intercept = -1/Vmax Not accuarate at extremes |
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Kcat
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Vmax=Kcat[etotal]
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Competitive Inhibition
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Increased KM, same vmax, reversible, ie methanol, anastrazole TIC
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Noncompetitive Inhibition
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Sam KM, lower Vmax, Irreversible, eg exemestane AMPSA
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Uncompetitive (rare)
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affinity for enzyme intermediate complex, KM decreases sometimes stabilizes the ES complex and vmax decreases, reversible
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TIC
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trimethoprim
ibprofen caffiene (blocks PDE) |
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AMPSA
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aspirin
Malathion-insectiside Penecillin-inhibit cell wall synthesis Sublactan Antabuse |
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Uncompetitive Inhibitor-
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Lithium
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An example of proteolytic regulation:
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digestion-eg trypsin activates chymotrypsin and starts a cascade, gets messed up by smoking because a meth binding site is oxidized and you end up with too much elactase that digests your aveolis
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Pegylation
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addition of polyethylene glycol PEg increases enzyme or peptide duration makes something more resistant to proteolytic regulation, some drugs use this
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Phosphorylation-
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more than 30% of enzymes are regulated by this, insulin receptor, covalent tightly regulated amplifiable, common target is serine
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Homotropic
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substrate is its own regulatory ie o2,
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Concerted versus sequential
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does it go from tense to relaxed all at once or does it change subunit by subunit
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Heterotropic
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different compounds ie H+ changes in reference to altitude-, you make more 2-3 bpg at a high altitude
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Isomeric-
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one binding site -nice hyperbolic curve
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Oligomeric
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When hill is not 1 there is cooperativity, n = hill coefficient, allosteric effects
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How much O2 saturation do you need and how much is in water?
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O2 saturation 2 ml/100ml in water, w/ hb it's 200/100 ml
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Which side of the tRNA is charged with the AA?
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3’ end acceptor stem
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What is a signal sequence, SRP, and SRP receptor and how do they affect translation?
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* Signal Sequence- present on most polypeptides targeted for membrane or secretion. Targets it to ER for translation and folding
* Signal Recognition Particle (SRP)- free floating protein that recognizes the signal sequence on a newly formed protein. SRP receptor- docking protein on the ER recognizes and bind the complex. * The polypeptide with the sequence is inserted through a hydrophobic pore in the ER membrane (translocon) * A signal peptidase usually removes the signal sequence (once inside the ER) * Once synthesis of the protein is complete, the protein is delivered to its cellular location. |
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How does protein know where to go?
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20 AA signal sequence (post translational mod?) on the newly forming protein
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What happens to protein before it leaves the ER? (two things)
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It is glycosylated and put in a vesicle
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What is the affect of acetylation on charge of a protein
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it neutralizes the charge of the amine group
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What turns off trypsin?
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Alpha 1 Antitrypsin (AAT)
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