Ch. 27 Protein Synthesis Flashcards
(97 cards)
1
Q
Where does the majority (90%) of the energy from a cell go?
A
It goes to protein synthesis.
2
Q
How many amino acids are there and does this match with the number of possible codons?
A
There are 20 amino acids, but there are 64 possible codons because each codon is three nts and there are four possible nts.
3
Q
What is a codon?
A
Three nucleotides that encode for a specific amino acid.
4
Q
What are the two main properties of codons?
A
They are non-overlapping and there are no gaps between codons.
5
Q
How many possible reading frames are there?
A
There are three possible reading frames depending on which nucleotide of the first three you start with. Only one is correct.
6
Q
How was the codon usage table made? (6)
A
- Have 20 tubes with a total homogenate and radioactively labeled poly(u)
- Incubate tubes at 37℃
- Wait for a radioactive protein to be made
- Separate protein with SDS-PAGE
- Only one tube gave a poly(u) radioactive band and it was phenylalanine (UUU)
- Repeat the procedure with poly(c), poly(a), and polu(g) to make codon usage table
7
Q
Why is the codon usage table such a great tool?
A
EVERYTHING uses the same codon usage table.
8
Q
What does it mean that the codon usage table is degenerate?
A
There is more than one codon for many amino acids.
9
Q
What does it mean that the codon usage table has wobble?
A
The third nucleotide isn’t always a perfect match and can vary.
10
Q
Why is wobble beneficial for protein synthesis? (2)
A
- Mutation in the wobble position may not matter and be less deleterious
- Protein synthesis is faster because only two nucleotides in the codon are complementary, so tRNA can leave easier
11
Q
What does the codon-anticodon interaction balance?
A
The codon-anticodon interaction is a balance between fidelity and speed.
12
Q
What are the five steps of protein synthesis?
A
- Activation of tRNA
- Initiation
- Elongation
- Termination
- Folding and Posttranslational Modification
13
Q
What generally happens during activation of tRNA?
A
A specific amino acid is covalently attached to a specific tRNA by aminoacyl-tRNA synthetase to produce an aminoacyl-tRNA. (there are 20)
14
Q
What is the ΔG of the activation of tRNA and why?
A
It is a positive ΔG because two phosphoanhydride bonds are broken.
15
Q
What generally happens during initiation?
A
The mRNA, small ribosomal subunit and the first aminoacyl-tRNA come together and then the large ribosomal subunit binds to all of these enclosing them.
16
Q
What does initiation require?
A
It burns one GTP.
17
Q
What generally happens during elongation?
A
The second aminoacyl-tRNA comes into the ribosome, an peptide bond forms, and the ribosome translocates. This process repeats over and over.
18
Q
What does elongation require?
A
It burns one GTP when the aminoacyl-tRNA comes into the ribosome and another when the ribosome translocates. So, two GTP are burned in total.
19
Q
What is translocation?
A
When the ribosome moves one codon down the mRNA.
20
Q
What generally happens during termination?
A
The ribosome hits a stop codon which doesn’t have an aminoacyl-tRNA, and the complex falls apart releasing the immature protein.
21
Q
What generally happens during folding and posttranslational modification?
A
Secondary and tertiary structure happen during translation, cofactoras are added, additional postranslational modifications are applied, and the protein is transported to its usage location.
22
Q
What are examples of cofactors and posttranslational modifications?
A
Cofactors: Zn, heme…
Posttranslational modifications: oligosaccharide side chains, proteolysis, methylation…
23
Q
What are ribosomes and what is their structure?
A
The machinery that carries out protein synthesis. It is made of two subunits, the 30S small subunit and the 50S large subunit that will come together to form the 70S ribosome.
24
Q
What are Svedberg units (S)?
A
They are units similar to density. It is measured with centrifugation and the more dense, the higher the S unit. They are NOT additive!
25
What is the main molecule in ribosomes and why is this unique?
The main molecule is rRNA. The rRNA does catalysis, so the proteins present are only there for structure, not cataytic ability.
26
What chemical property does rRNA have and what does it resemble?
rRNA has significant complementary H-bonding with itself and can fold back on itself resembling protein structure. (Very Specific!)
27
How do ribosomes assemble?
Ribosomes self assemble.
28
What is tRNA?
The molecule responsible for translating nucleic acid language to protein language.
29
How many tRNAs are there and how are they similar (2)? Different (1)?
There are 20 tRNAs. They are all very similar in sequence and end in .....CCA-3'. But, several bases are chemically modified on the tRNAs.
30
Where is the amino acid attached to a tRNA?
The amino acid is attached to the adenine on the 3' end. ("amino acid arm")
31
Other than an amino acid, what else does a tRNA contain?
It has an anticodon that complementary H-bonds with the codon.
32
Where does activation and initiation take place?
Both take place in the cytosol.
33
What are the steps of activation of tRNA? (3)
1. ATP is covalently attached to the amino acid
2. two phosphoanhydride bonds of ATP are hydrolyzed
3. the amino acid is connected at the 3'OH of the tRNA's adenine to make a charged tRNA
34
What happens if an amino acid is added to the 2'OH of a tRNA's adenine instead?
It will be isomerized to end up on the 3'OH.
35
Can activation of tRNA be proofread?
There is some proofreading, but mistakes still occur.
36
Why is it okay that there are some mistakes in tRNA activation? (~3)
There are lots of copies made, it is only one amino acid out of many, and there is lots of protein degradation, so fidelity doesn't take a hit.
37
What is always the start codon and how is it special?
AUG (Met) is always the start codon. The start Met has its own tRNA, and normal Met has a different tRNA.
38
What are the steps of initiation? (5)
1. small subunit binds IF1 and IF3 (IF3 blocks the large subunit from binding)
2. small subunit binds mRNA
3. charged tRNA (Met tRNA) binds the P site
4. IF2 binds the complex (IF2 must be bound to GTP first)
5. large subunit binds the complex, completing the ribosome, and IF's fall off after IF2 hydrolyzes GTP→GDP
39
How does the small subunit know where to bind the mRNA?
The Shine Delgarno Sequence tells the ribosome where to bind and the mRNA is held in perfect orientation (AUG in P site) with in the cleft.
40
What is the Shine-Delgarno Sequence?
A homologous sequence just upstream of the start codon that H-bonds with specific parts of rRNA in a ribosome.
41
What are the three sites on the small ribosomal subunit and define/name each.
A Site (Aminoacyl Site): where fresh charged tRNAs come in
P Site (Peptidyl Site): where peptide bond formation occurs
E Site (Exit/Eject Site): where uncharged tRNAs leave
42
What are the steps of elongation? (8)
1. EF-tu must be bound to GTP, then it binds an aminoacyl-tRNA and enters the A site
2. EF-tu hydrolyzes GTP causing a conformation change that leads to dissociation between EF-tu and aminoacyl-tRNA
3. EF-ts binds EF-tu and helps it reload (GDP→GTP) and get ready bind the next aminoacyl-tRNA
4. Ribosome hold the amino acids in perfect orientation (amino group next to carbonyl carbon) for peptide bond formation
5. Peptidyl transferase catalyzed the formation of a peptide bond and cleavage of the ester bond between the a.a in the P site and its tRNA
6. Ribosome translocates due to EF-G bound to GTP in the A site (uncharged start tRNA in the E site, protein-tRNA in the A site, next tRNA in the A site)
7. EF-G hydrolyzes GTP in the A site causeing a conformation change that leads to dissociation of EF-G and the A site (empty A site)
8. Start from the top with the next aminoacyl-tRNA
43
How many phosphoanhydride bonds does one amino acid addition cost?
Each amino acid in a protein costs FOUR phosphoanhydride bonds!
44
What are the steps of termination? (5)
1. ribosome reaches a stop codon (no tRNA)
2. release factor (RF) binds in the A site
3. peptidyl transferase cleaves the bond between the tRNA in the P site and its amino acid releasing the protein
4. RF helps the ribosome fall apart
5. IF3 binds the E site of the small ribosomal subunit to make it ready to restart
45
When do cells do protein synthesis?
They ONLY do protein synthesis when they have to because it is extremely energy intensive.
46
What is a polyribosome?
One mRNA chain that has many ribosomes translating to make more protein copies in less time.
47
What is the only limit to polyribosome translation?
mRNA half life is the only limit.
48
How do polyribosomes form?
As soon as one ribosome translocates, another can bind.
49
When does protein folding happen?
It happens almost immediately after the polypeptide is created.
50
What are examples of N-terminal capping? (3)
A posttranslational modification where the N-terminus can be acetylated (add 2C), signal sequences (pre- and pro-) cleaved, or the start Met cleaved.
51
What direction does protein synthesis occur?
N-terminus to C-terminus. Always add to the C-terminus of a protein.
52
What is protein phosphorylation?
A posttranslational modification where a phosphate group is added to an amino acid with a -OH group.
53
What are oligosaccharide side chains?
They are posttranslational modifications where oligosaccharides are attached to proteins in the ER .
54
Which proteins are on the surface of the ER? (2)
Membrane-bound proteins and secreted proteins are on the surface of the ER.
55
Where does protein glycosylation occur and what is it?
It is a posttranslational modification that signals a protein is destined for the cell surface or secretion. it starts in the ER and finishes in the Golgi.
56
What are the two types of protein glycosylation?
O-linked: linked via an oxygen of serine or threonine
N-linked: linked via the nitrogen of an asparagine
57
What are disulfide bonds?
A posttranslational modification that forms a bond between cysteine residues. They can be interchain or intrachain.
58
What are lipid anchors?
A posttranslational modification where lipids are added to proteins for recruitment to the face of the plasma membrane. (e.g. Ras and Gɑ)
59
What are zymogens?
Proteins that are synthesized as inactive precursors and have a few amino acids proteolytically cleaved ot become active. A posttranslational modification. (e.g. pepsinogen)
60
What are toxins?
They are small molecules synthesized by organisms to inhibit protein synthesis in other organisms as a defense mechanism.
61
How do humans take advantage of toxins?
We have lots of antibiotics that work in a similar way.
62
What is tetracycline?
A toxin that binds to the A site of bacterial ribosomes and blocks EF-tu and tRNA from binding the A site.
63
What is diptheria?
A strain of bacteria that secretes diptheria toxin.
64
What is diptheria toxin?
A toxin that causes ADP-ribosylation: covalently attaches ADP to EF-2 which blocks elongation in human protein synthesis.
65
What is ricin?
A toxin that covalently attaches to a nucleotide in the large ribosomal subunit rRNA blocking protein synthesis.
66
How can ricin be made?
It is easy to isolate from caster beans. Just do an aqueous extraction.
67
What is targeting?
Transporting proteins from where they are synthesized (cytosol) to where they will become resident.
68
What are signal sequences?
A short a.a. squence on the N-terminus of a protein that acts as an address label to get the protein where it needs to go. (e.g. "ADEL" goes to the ER forever)
69
What does it mean that the ER is in a constant state of flux?
In addition to being the site of synthesis for many lipids, there are also many proteins synthesized on the face of the ER that then bleb to the Golgi. "Bulk Flow Model"
70
What happens during translation if a protein has a signal sequence? (7)
1. When the signal sequence exits the ribosome it will be bound by signal recognition protein (SRP)
2. SRP binding to the signal sequence causes protein synthesis to pause
3. The whole complex docks on face of the ER
4. SRP is bound by peptide recognition complex: integral membrane protein in the ER
5. SRP hydrolyzes GTP causing a conformation change and SRP leaves
6. Protein synthesis resumes
7. Signal sequence is clipped off by an ER protease
71
Where will soluble proteins be translated?
Soluble proteins are translated directly into the lumen of the ER.
72
Where will integral membrane proteins be translated?
Integral membrane proteins will be translated with the transmembrane domain embedded into the ER membrane.
73
What kind of protein does the ER contain a lot of?
The ER is home to many chaperone proteins that help proteins fold correctly.
74
How does N-linked glycosylation occur? (7)
1. 2 GlcNAc (carbohydrates) get covalently attached to dolichol phosphate
2. 5 mannoses are added to dolichol phosphate in two branches
3. the whole molecule gets inverted
4. 4 more mannoses get added to dolichol phosphate (now 9 in total)
5. 3 glucose residues added to dolichol phosphate
6. oligosaccharide is clipped off of dolichol phosphate and transferred to the asparagine residue of the target protein
7. almost all glucose and mannose residues get removes and replaced by individual carbohydrates (in Golgi!)
75
What recognize oligosaccharide side chains?
Lectins bind and recognize specific oligosaccharide side chains.
76
What is the general movement from ER to Golgi?
ER blebs to cis Golgi, cis Golgi blebs to medial Golgi, medial Golgi blebs to trans Golgi.
77
What happens to the luminal contents of the ER when it moves to the Golgi? Membrane contents?
The lumen of the ER will remain in the lumen of the Golgi. Things embedded in the ER membrane will remain in the Golgi membrane.
78
What happens to soluble proteins in the Golgi?
Proteins that are soluble in the Golgi lumen will be secreted.
79
What happens to proteins in the Golgi membrane?
Proteins that are in the Golgi membrane will to go the plasma membrane.
80
Do all membrane proteins leaving the Golgi fuse with the plasma membrane?
No. Some vesicles don't fuse with the plasma membrane, they stay just inside (like GLUT4 and insulin).
81
What is endocytosis?
"bringing things into the cell"
82
What are the steps of endocytosis? (6)
1. must have a receptor interact with a ligand
2. when the ligand is bound by a receptor, clathrin starts to polymerize
3. as clathrin starts to polymerize, the plasma membrane will invaginate
4. dynamin seals off the vesicle/endosome
5. clathrin falls off the endosome
6. membranes and receptors will bleb off adn go back to the p.m. while the lumen will fuse and mix with other endosomes
83
What is clathrin?
An integral membrane protein with six subunits that have a soccer ball-like structure that causes a "pit"/invagination.
84
What kind of pump does an endosome have?
Vesicles have F₀F₁ ATPases that run in reverse and hydrolyze ATP to pump protons into the vesicle.
85
What do the pumps on the endosome cause?
Protons are pumped into the vesicle which lowers the pH. The low pH causes a conformation change and receptors no longer bind ligands.
86
What kind of proteins are endosome receptors? Ligands?
Receptors are integral membrane proteins. Ligands must be soluble proteins.
87
What is caveolin?
Another protein that works basically the same as clathrin.
88
Why are proteins degraded?
Proteins get old and can spontaneously misfold, or they are just no longer needed.
89
What is a typical protein half life?
It can be minutes or days.
90
Why are old proteins a problem?
Old proteins can form a plaque and kill cells. This is seen in conditions like Parkinson's or Alzheimer's, but does not happen in healthy cells.
91
What is ubiquitin?
A peptide that is found EVERYWHERE and covalently attached to lysines of misfolded or unneeded proteins.
92
What does ubiquination require?
It requires 3 ATP.
93
What is polyubiquination?
When the lysine of ubiquitin is unbiquinated, so on and so forth.
94
Why would have more than one ubiquitin on a protein?
The more ubiquitin, the faster the degradation.
95
What is the fate of a protein tagged with ubiquitin?
Proteins tagged with ubiquitin are degraded by the 26S proteasome.
96
What are the steps of protein degradation in the 26S proteasome? (3)
1. proteasome binds ubiquitin and drags the protein in removing 2°, 3°, and 4° structure
2. breaks all/nearly all peptide bonds nonspecifically using ATP hydrolysis
3. amino acids are released out the bottom
97
What feature of a protein plays into its half-life?
The amino-terminal amino acid matters for protein half-life.
