What is the genetic code?
A set of 64 codons (triplet of nucleotides) that each code for one amino acid (some amino acids have multiple codons)
What does it mean to say that the genetic code is degenerate?
Most amino acids are represented by more than one codon
Degenerate codons tend to contain the same nucleotides in the ___ positions and vary in the ___ positions.
First and second; third (Wobble)
What is translation?
The process of translating mRNA into protein
True or false - the genetic code varies for different organisms.
False - the genetic code is universal in all organisms.
An mRNA consisting of contiguous triplet codons can be read in three different ___. How many are correct?
Frames; only one.
What are the 4 basic steps of translation?
1. Charging of the tRNA 2. Initiation 3. Elongation 4. Termination
Each ___ is specific for an amino acid.
What synthesizes tRNA?
RNA Polymerase III
What is the anticodon?
A sequence of three nucleotides in the tRNA that base pairs with a codon in mRNA
tRNA is modified after synthesis; why?
Helps maintain its folding
Once the first two positions are paired, the exact pairing of the third position is less critical. This is the ___ position. What is the purpose of this?
Wobble; permits some tRNAs to recognize more than 1 codon.
For each wobble base codon, what are the possible anticodon bases? (U, C, A, G)
U -> A, G, I C -> G, I A -> U G -> C
If referring to the anticodon, the Wobble position is the ___ base. If referring to the codon, the Wobble position is the ___ base.
What catalyzes the two-step activation of a t-RNA?
Draw the two-step activation of a t-RNA.
The genetic code is translated by means of two adaptors that act one after another. What are these and what do they do?
1. Aminoacyl-tRNA synthetase - couples the amino acid to the corresponding tRNA
2. tRNA - its anticodon base pairs with a codon on the mRNA
How does the aminoacyl tRNA synthetase recognize the correct tRNA?
Extensive structural and chemical complimentarity; three adjacent binding pockets in the synthetase match the shape and charge of the nucleotides in the anticodon.
How does the aminoactyl tRNA synthetase select the correct amino acid?
Proofreading activity via hydrolytic editing - an incorrect amino acid fits into the editing site and is cleaved. A correct amino acid does not fit, confirming that it is correct. The correct aminio acid has the highest affinity for the synthesis site (versus the editing site).
The proofreading activity of aminoacyl-tRNA synthetase is very accurate; there is one mistake in ___ couplings.
Describe the process by which prokaryotes select the correct reading frame for protein synthesis.
Shine-Dalgarno sequences are located ~10 nucleotides upstream of the initiator codon in mRNA. These sequences are complementary to the 3' end of the 16S-ribosomal RNA in the prokaryotic 30S subunit. The ribosome is positioned for translation initiation in the correct reading frame by base pairing between the 16S rRNA and Shine-Dalgarno sequences.
What are Shine-Dalgarno sequences?
Purine-rich sequences of 3-9 bases that bind the ribosome in prokaryotes in order to set the correct reading frame for translation.
How do eukaryotes select the correct reading frame?
eIF2 sets the frame; AUG start codon
What are the steps to initiation of protein synthesis in prokaryotes?
1. The 30S subunit base pairs with the SD sequence upstream of the AUG start codon. This positions the initiating codon in the P site of the ribosome. 30S binds IF-3, preventing premature assembly with 50S.
2. A ternary complex between IF-2, GTP, and fMet-initiator tRNA binds in the P site of 30S, permitting base-pairing between mRNA initiation codon and fMet-tRNA (methionine-formyl methionine) anticodon.
3. The large complex formed in step 2 binds 50S subunit. GTP hydrolysis occurs and GDP, inorganic phosphate, IF-2, and IF-3 dissociate.
What do the subunits of the ribosome do?
The small ribosomal subunit accurately matches the codon and anti-codon. The large ribosomal subunit catalyzes peptide bond formation.
What are the steps to initiation of protein synthesis in eukaryotes?
1. Initiator tRNA (with bound eIF2-GTP) is loaded into the small ribosomal subunit at the P site.
2. eIF4E and eIF4G bind to the 5' cap and the polyA tail of the mRNA, ensuring the mRNA is not broken. This aids recruitment of the 40S subunit (which has eIF2-GTP*met-tRNA), which binds to the mRNA at the 5' end.
3. eIF-4A and 4B facilitate the unwinding of the mRNA secondary structure so 40S can scan the untranslated region until a suitable AUG is reached. ATP hydrolysis catalyzes this step.
4. eIF2-GTP is hydrolyzed, eIFs dissociate, and the large 60S ribosomal subunit binds to the 40S. Synthesis is ready to begin.
What is the rate limiting step of translation initiation in eukaryotes and why?
The cleavage of eIF2-GTP to GDP; eIF2-GDP must be recycled to eIF2-GTP (catalyzed by eIF2B)
What are the steps to elongation in protein synthesis in eukaryotes?
1. Ribosomes contain 4 binding sites - mRNA site, A-site, P-site, and E-site. At the end of initiation, the initiation tRNA is in the P-site. An aminoacyl-tRNA molecule binds to a vacant A-site.
2. A new peptide bond is formed between the amino acids in A and P.
3. Large subunit translocates relative to the small subunit, leaving the 2 tRNA in hybrid sites (large in P, small in A, and large in E, small in P). This is catalyzed in part by the peptidyl transferase activity of the large subunit.
4. Small subunit translocates, carrying the mRNA 3 nucleotides and resetting the ribosome
In what direction does translation occur?
5' to 3'; N-term end of the protein is created first
Elongation requires what two things?
GTP hydrolysis and the participation of 3 elongation factors, including 2 GTP-binding proteins
What are the two major elongation factors in prokaryotes? In eukaryotes?
Prokaryotes: EF-Tu and EF-G
Eukaryotes: EF-1 and EF-2
What do EF-Tu and EF-G do?
1. EF-Tu*GTP binds to a charged tRNA as it enters the ribosome. GTP hydrolysis and the exit of EF-Tu cause 2 short lags that provide an opportunity for an incorrect tRNA to exit prior to incorporation.
2. EF-G*GTP binds near the A-site and accelerates the movement of tRNAs to the P and E sites. GTP hydrolysis catalyzes conformational changes needed to move tRNA and and advance the mRNA.
What catalyzes reactivation of Ef-TU?
What are the steps of translation termination?
1. Stop codon is reached.
2. Release factor eRF1 binds to the ribosome's A site and forces peptidyl transferase to catalyze addition of water instead of an amino acid.
3. Frees carboxyl terminus; protein released
4. Ribosome dissociates into 2 separate units
Why can eRF1 bind to the A site?
It mimics tRNA structurally and chemically
What 5 steps contribute to the fidelity of protein synthesis?
1. Aminoacyl tRNA synthetase must recognize correct tRNA.
2. Aminoacyl tRNA synthetase must select the correct amino acid.
3. mRNA must be fully processed (in eukaryotes) prior to translation initiation.
4. The ribosome matches the mRNA codon with the tRNA anticodon.
5. GTP hydrolysis and release of EF-Tu elongation factor provide short lags the allow incorrect tRNA to be released before irreversible addition.
Compare and contrast the site of translation in prokaryotes and eukaryotes.
Prokaryotes: Transcription and translation are coupled; protein synthesis can begin before mRNA synthesis is done (no processing)
Eukaryotes: pre-mRNA is processed in the nucleus, mature message exported to the cytoplasm where translation occurs
Prokaryotes are polycistronic; eukaryotes are monocistronic. What does this mean?
Prokaryotes have multiple SD ribosome binding sites and each can synthesize a different protein at the same time. Eukaryotes can only synthesize one protein at a time.
Compare and contrast the ribosome structures of prokaryotes and eukaryotes.
Prokaryotes: 70S (50S - large, 30S - small)
Eukaryotes: 80S (60S - large, 40S - small)
Compare and contrast prokaryotic and eukaryotic sensitivity to antibiotics.
Inhibitors can act only on eukaryotes, on both eukaryotes and prokaryotes, or on prokaryotes only.
What is puromycin?
An antibiotic that causes premature release of nascent polypeptide chains by its addition to the growing chain end; acts on both prokaryotes and eukaryotes. It is also an example of molecular mimicry - it looks like tRNA and halts protein synthesis by binding to the ribosome.
What are the 4 steps required to create a functional protein?
2. Cofactor binding
3. Covalent modifications
4. Assemble with partner proteins
True or false - proteins can begin to fold as they are synthesized.
Which end of the protein folds first?
Some proteins requires assistance from chaperones to fold. What are two families of chaperones and what do they do?
1. Hsp70 family: act early, recognize small stretches of hydrophobic amino acids on the surface of a protein; ATP-bound Hsp70 grabs the taret and hydrolyzes ATP to ADP. Conformational changes occur that cause tighter association of hsp70. Repeats this cycle to help protein to fold.
2. Hsp60 family: forms barrel to isolate misfolded proteins, prevent aggregation, and provide a favorable environment to refold. Captures the protein via hydrophobic amino acids on the surface.