Translation Flashcards
What are the stop codons?
UAA
UGA
UAG
What is the start codon and what does it code for?
It is AUG and it codes for methionine.
What is a pneumonic for UAA?
You are angry
What is a pneumonic for UGA?
You go away
What is a pneumonic for UAG?
You are gone
What does UUU code for?
Phenylalanine
What does CCC code for?
Proline
What does GGG code for?
Glycine
What does AAA code for?
Lysine
What is the in-frame sequence between the initiation codon and the termination codon called?
The open reading frame (ORF)
What does the N-terminal of protein correspond to on mRNA?
The 5’ end
What does the C-terminal of protein correspond to on mRNA?
The 3’ end
What did Dintzis do in 1961?
Used radioisotopes to follow globin synthesis in reticulocytes (immature erythrocytes)
Can RNA interact directly with amino acids to specify their order?
Nope, tRNAs serve as adapters specific for each amino acid that bind to specific codons on RNA templates
What happens to aa’s prior to their incorporation into protein?
They are enzymatically linked to tRNA molecules
Tell me 4 basic things about tRNA.
Small RNA molecules (73-93 bases)
Mitochondria have their own
All cells have at least 32 tRNAs
Some tRNAs can recognize more than one codon
What do most tRNAs have at their 5’ end?
G
How many bases are often modified by methylation of a normal base in tRNAs?
8 or more
What does the D arm of tRNA contain?
Dihydrouride
What is the anticodon of tRNA structure?
A 3 base structure in the anticodon loop on the anticodon arm
What does the anticodon of tRNA do?
Pairs with the codon in the mRNA
What is the first base of the anticodon called?
The wobble position
What are the 4 relationships between codons and anticodons as outlined by the wobble hypothesis?
1) First two bases of codon always form strong standard base pairs
2) The first base of some anticodons determines the number of codons read by a given tRNA
3) Different tRNAs are required when codons differ in either of the first two bases
4) A minimum of 32 different tRNAs to translate all 61 codons
What is an example of the wobble hypothesis?
Yeast arginine tRNA
What are ribosomes composed of?
Two subunits one small one large
Are ribosomes large or small?
Large! Largest at 23nm
What do both subunits of ribosomes form?
Peptidyl (P) site
Aminoacyl (A) site
Exit (E) site
What are the ribosome sizes in prokaryotes?
Large: 50s, small: 30s, ribosome: 70s
What RNA are used in prokaryotes?
Large uses 23s and 5s RNA. Small uses 16s RNA
What are the 5 major stages of protein synthesis?
Activation of amino acids Initiation Elongation Termination and ribosome recycling Folding and post translational processing
How are tRNAs charged/activated?
Amino acids are attached via ester bonds to the 3’OH of the corresponding tRNA (aminoacylation)
What catalyzes the charging of tRNA?
Aminoacyl-tRNA synthetases (specific to aa’s and tRNAs)
What are the two steps in the enzyme active site for tRNA charging?
1) An enzyme bound intermediate is formed by the reaction of ATP and amino acid (hydrolysis of ppi)
2) Aminoacyl group transferred to corresponding specific tRNA
What makes tRNA charging irreversible?
The hydrolysis of ppi because two energy rich bonds are used
Is the identity of the aa attached to the tRNA checked by the ribosome?
No, but some aa-tRNA synthetases proofread
What are some ways that Aminoacyl-tRNA synthetases proofread?
Some recognize anticodon itself
Some recognize 10 or more bases in tRNA
Some recognize one specific base pair
What makes the tRNA ready to bind to the ribosome a site?
Aminoacyl-tRNA form a complex with EF-Tu that also binds one GTP molecule.
How many tRNAs for methionine do all organisms have?
2; one exclusively for initiation, the other for methionines within the polypeptide chain
What is the starting amino acid in prokaryotes?
N-formylmethionine
What are the 7 components required for initiation in bacteria?
30S ribosomal subunit mRNA Initiation fmet-tRNA^fmet Three initiation factors GTP 50S ribosomal subunit Mg2+
How does the small 30S subunit position itself on mRNA template in step 1 of bacterial translation initiation?
Codon is bound in a region of 30S subunit that is part of the P site
The initiation codon is distinguished by proximity to Shine-Dalgarno sequence
What does IF-1 block?
The A site
What does IF-3 do?
Prevents the 30S and 50S subunits from combining prematurely
How does the initiator tRNA bind to the P site in step 2 of bacterial translation initiation?
The 30S subunit, mRNA, IF-1, IF-3 complex is joined by IF-2 (itself bound to GTP) and fmet-trna^fmet
Where does the anticodon of the initiator tRNA pair with the initiation codon?
Only in the P site (fmet-trna^fmet will only bind to the P site)
How does the large 50S subunit bind to the complex in step 3 or bacterial translation initiation?
The large complex binds the 50S ribosomal subunit resulting in GTP bound by IF-2 hydrolyzed to GDP which is then released and all intrinsic factors leave the complex
What are the components of the final initiation complex?
Charged initiator tRNA in complete P site and an empty A site
How is initiation in eukaryotes different?
At least 9 initiation factors involved
One binds to 5’ cap and complex formed with 40S subunit
Kozak sequence found at many initiation sites
If ORF is short, translation will begin again at next AUG
Complete 80S ribosome assembled using other IFs and initiating Met-trna^met
What is elongation?
The stepwise addition of aa’s to polypeptide chain by formation of peptide bonds.
In both prokaryotes and eukaryotes how many ribosomes are found bound to each mRNA molecule?
10-100 ribosomes
How does elongation allow efficient use if mRNA?
Many polypeptides can be synthesized simultaneously using just one molecules worth of genetic information.
What does elongation require in bacteria?
Initiation complex (for first peptide bond) Next Aminoacyl-tRNA (specified by next codon) Elongation factors (EF-Tu, EF-Ts, EF-G)
What are the 3 steps of elongation in bacterial translation?
1) Bind next Aminoacyl-tRNA
2) Peptide bond formation
3) Translocation
What enzyme catalyzes peptide bond formation in elongation in bacterial translation?
Peptides transferase which is a ribozyme
Where does the energy for bacterial translation elongation come from?
From the high energy ester bond between aa and tRNA
Where do the spent tRNAs shift to?
They shift from the P site on the 30S to the E
After the spent tRNA move what happens?
The A site is now empty, ready for the next aa-tRNA
Can the A and E sites be occupied simultaneously?
Nope
Why are two steps used to move tRNAs?
Anchors one end of tRNA while other moves during peptide bond formation and translocation
Prevents premature release of peptidyl-tRNA (waste of energy) and enhances processivity
What is proof reading on chromosome limited to?
Codon-anticodon interactions
While GTP and GDP are bound base pairing is checked, incorrect base pairs are dissociated
What is elongation proof reading optimized for?
Speed and fidelity: slower speeds=higher fidelity and vice versa
How many termination factors are there?
3 in prokaryotes (RF1, RF2, RF3)
1 in eukaryotes (eRF)
What stop codons bind to RF1?
UAG UAA
What stop codons bind to RF2?
UGA UAA
How are transcription and translation tightly coupled in bacteria?
Protein synthesis actually takes place before mRNA completed or degraded
How expensive is protein synthesis?
At least 4 high energy bonds expended per peptide bond
122 kj/mol for one 21kj/mol peptide bond
Why is protein synthesis so energy costly?
To maintain proper alignment of codon, tRNA and growing end of polypeptide chain
What percent of large proteins (1000aa) are synthesized correctly?
90%
At what rate are smaller proteins synthesized correctly?
99%
