Lecture 5B - Translation Flashcards
How important is translation from an energy standpoint?
Cells devote more energy to protein synthesis than any other aspect of metabolism
How do four DNA bases encode the sequence of proteins that contain 20 different amino acids?
Using triplet code (codon)
4^2 = 16 AA, but 4^3 = 64 AA
What is evidence for triplet code?
Method: in vitro translation system (isolated translational components)
- Synthetic trinucleotides are sufficient to stimulate binding of aminoacyl-tRNAs to ribosomes
- Synthetic dinucleotide repeating mRNAs direct synthesis of polypeptides w/ 2 different AA
3, Synthetic trinucleotide repeating mRNAs direct synthesis of three different polypeptides w/ each containing the same AA
Triplicate codon sequences must be read in the _______ ________ ________, otherwise frame shift occurs => nonfunctional errors
Correct reading frame
7 properties of the genetic code
- Nucleotide is triplet based (Codon)
- Code is degenerate or redundant
- Code is non-overlapping
- Code is comma-free
- Code is ordered
- Code contains start and stop codons
- Code is nearly universal
What does it mean to say code is degenerate/redundant? What does this mean for tRNA?
Several codons encode the same AA; all but 2 AA are encoded by more than 1 codon
tRNAs can bp w/ more than 1 codon or there is more than one tRNA for many AA (both true)
What does it mean to say code is non-overlapping?
Nucleotides belong to only one codon
What does it mean to say code is comma-free?
Codons are read consecutively
What does it mean to say code is ordered?
Codons for same AA are similar, and codons for chemically related AA are similar
What does it mean to say codon contains start and stop codons?
AUG: start codon
UAG, UAA, UGA: stop codons
What does it mean to say that code is nearly universal?
Codons have the same meaning in all organisms
What is Crick’s wobble hypothesis?
- Base-pairing rules only apply for the first two nucleotides of the codon
- Base-pairing of the third nucleotide of codon is less stringent => this site can wobble
- Inosine on tRNA can base pair w/ uracil, cytosine, or adenine
- Allows for degenerate, but ordered genetic code
Describe AA activation/tRNA “charging”; Where does the AA bind? How many aminoacyl tRNA synthetases?
- AA is adenylated
- Aminoacyl tRNA synthetase catalyzes reaction b/t adenylated AA and tRNA to make AMP and aminoacyl-tRNA, matching the correct AA for the correct tRNA
AA binds at carboxyl carbon to 3’ OH of tRNA ribose
There is 1 aminoacyl tRNA synthetase (30-40) for each AA
What is hydrolytic editing of tRNA synthetase?
A trial by error mechanism - if AA is incorrect, it fits into the editing site and is cleaved from tRNA
Similar to DNA pol
Know the sizes (in S units) of euk and prok intact ribosomes and ribosomal subunits (small & large); What does the S mean?; Why is total different from subunit S?; which subunit binds to mRNA first?
Prokaryotic ribosome sizes: 30S + 50S = 70S
Eukaryotic ribosome sizes: 40s + 60S = 80S
S is Svedberg unit
Total is different due to sedimentation during centrifugation)
Small subunit binds mRNA first
Sites for tRNA binding to ribosome; how does ribosome move along mRNA
A-site: binds incoming aminoacyl tRNA
P-site: site of incorporation of new AA to growing polypeptide chain; binding site for peptidyl-tRNA
E-site: binding site for tRNA release after growing polypeptide chain has been transferred to aminoacyl-tRNA
Ribosome moves via translocation
What catalyzes peptide bond formation in ribosome (proks vs euks)? Which subunit moves first?
Peptidyl transferase, a part of large ribosomal subunit (23S in proks, 28S in euks)
Large subunit moves first, followed by small subunit
(refer to slide 28, lec 5B)
Ribosome composition by mass relative to rRNA and proteins?
Ribosome is 50/50 rRNA and ribosomal proteins
Ribosomes have how many proteins and rRNAs?
> 50 proteins and 4 rRNAs
Where are ribosomes located in proks vs euks? What happens to ribosome subunits after translation?
Proks: located throughout cell
Euks: located in cytoplasm, frequently on rough ER
After translation, subunits dissociate
What are tRNAs in the context of translation machinery? How many versions?
Adaptor molecules that deliver amino acids to ribosome where it H-bonds to mRNA template
40-60 different versions
Ribosomes undergo _____ and ______ with each round of protein synthesis
Association; dissociation
What 4 things comprise the complex translation machinery?
- Ribosomes
- tRNAs
- AA activating enzymes (aminoacyl tRNA synthetase)
- Numerous other proteins involved in: initiation, elongation, termination
Eukaryotic vs Prokaryotic translation initiation
- Initiator AA is methionine but not formylated
- Proks have formylated methioinne - Initiator complex forms at 5’ end of mRNA (recognizes 7-MG cap and reads until it hits AUG start) vs prokaryotic Shine-Dalgarno start site
What is the significance of a formylated methionine initiator AA in prokaryotes?
The N terminus is capped via attachment of a 1C formate group => prevent unintended addition of polypeptide to that initiator AA
Eukaryotic initiation details
- The 7-MG cap on 5’ end of mRNA bound by cap-binding protein
- Other initiation factors bind CBP followed by 40S subunit
- Methionyl-tRNA (initiator AA) interacts w/ soluble initiation factors and binds ribosomal P site
- Complex moves 5’ to 3’ on mRNA searching for start codon
- Once at start codon => initiation factors fall off and 60S subunit binds Met-tRNA:mRNA:40S complex
Purpose of Cap-binding protein
Helps ribosome determine if mRNA is undamaged and allows small subunit scanning of AUG start to being
3 Steps of Chain Elongation
Same in proks and euks
- Aminoacyl-tRNA binds to A site
- Elongation factor Tu (EF-TU) + GTP - Peptide bond formation
- Peptidyl transferase activity of 23S rRNA in proks, 28S rRNA in euks + GTP - Translocation of ribosome to next codon
- EF-G + GTP
Function of EF-Tu
Escorts aminoacyl-tRNA to A site
- Charged tRNA bound to EF-Tu interacts w/ A site
- When correct codon-anticodon interaction occurs, EF-Tu binds to factor-binding center
-EF-Tu hydrolyzes bound GTP and is released
Function of EF-G, what is ribosomal translocation
Stimulates ribosome translocation
Translocation: for each round of peptide bond formation
- P-site tRNA moves to E site
- A site tRNA moves to P site
- mRNA moves by 3 nucleotides to expose next codon
- EF-G hydrolyzes GTP => facilitates translocation
Translational Termination
- Sequence specific (UAA, UAG, or UGA)
- When stop codons enter A site => Release factors enter and alter peptidyl transferase activity => H2O added to COOH terminus => releases peptide chain
Initiation factors, elongation factors, and release factors
IFs: bind CBP following 40S (small) subunit, interact w/ Met-tRNA to bind P-site, fall off once at start codon
EFs: EF-Tu and EF-G
RFs: enter A site with stop codons, altering peptidyl transferase activity and adding water to COOH terminus => release peptide chain
