Translation 02 Flashcards
(31 cards)
What is Aminoacyl tRNA synthetase?
Enzyme that catalyzes the esterification of a specific amino acid to its compatible tRNA to form an aminoacyl-tRNA.
Where does the amino acid connect with the tRNA?
tRNA 3’ end -phosphate-ribose/adenine - AA attached to 2’ or 3’ position of the ribose.
What is synthetase proof-reading activity?
It can selectively edit the incorrect (non-cognate) amino acid at the enzyme proof reading site. It has an editing site and a synthesis site .
What must happen to amino acid before binding to tRNA?
It must be adenylated (aa-P-ribose-adenine)
What are the 2 levels of control to ensure that the proper amino acid is incorporate into protein?
1) Charging of proper tRNA: by tRNA synthetase
2) Matching the cognate tRNA to the mRNA: based on the correct base-pairing of tRNA anticodon and mRNA codon.
What are the two methionine tRNAs?
- Initiator methionine tRNA: ensures initiation of translation. Enters ribosomal P site with initiation factors.
- Elongator methionine tRNA: incorporates internal methionines. Carried by elongation factors to A site.
*Both aminoacylated by methyonyl-tRNA synthetase. Synthetase distinguishes the two.
What is the Shine-Dalgarno sequencw?
It is a ribosomal binding site in mRNA, 8 bases upstream of the start codon AUG. Exists in bacteria and archaea, and also present in some chloroplastic and mitochondial transcripts
What are the initiation factors?
IF-1: occludes A-site
IF-2: small GTP bp, binds initiator tRNA and helps it dock with 30S
IF-3: helps reposition complex to properly math codon to anticodon; binds 30S to free it from complex.
What are the elongation factors?
- EF-Tu, EF-G: small GTP binding proteins
- EF-Ts: guanine nucleotide exchange factor for EF-Tu catalyzing the release of GDP. This enables EF-Tu to bind to a new GTP molecule
What happens during elongation?
- EF-Tu-GTP delivers aminoacyl-tRNA to the A-site (EF-Tu recognizes all aminoacyl-tRNAs)
- Empty tRNA in E-site exits complex
- A ribosome domain functions as GAP for EF-Tu
- GTP is hydrolyzed & incorrectly base-paired tRNAs dissociate. EF-Tu dissociates.
- tRNA accommodation (conformation relaxes, & acceptor stem is repositioned for catalysis)
- Peptidyl transferase in 50S ribosome transfers growing polypeptide to newest tRNA in A-site
- Translocation
What does the 16S rRNA interact with?
- Shine-Dalgarno sequence
2. Minor groove of the first two bp of the double helical codon/anticodon complex
What is the proof-reading mechanism in translation?
16S interaction with minor groove of codon-anticodon complex stabilizes a particular ribosomal conformation. IF this conformation is not generated, there is release of the aminoacyl-tRNA prior to peptide formation.
What is the 23S rRNA?
It can be considered a ribozyme. Transpeptidation (peptide bond formation) involves acid/base catalysis by an adenosine in the 23S rRNA of the large ribosomal subunit.
Where is the protein exit tunnel?
It is located midway between A and P site.
What the factors in terminations?
Release factors:
- RF1, RF2: recognize and bind the stop codons
- RF-3-GTP: facilitates binding of RF-1 or RF-2 to ribosome
What happens during termination?
- Stop codon in A site: UAG, UAA, UGA
- No EF-Tu-aa-tRNA complex is bound
- A release factor binds ribosome and activates peptdyl transferease
- Peptidyl transferase catalyzes transfer of the peptifyl group to water releasing free peptide.
- Hydrolysis of GTP on RF-3 results in dissociation of release factors
- Disassembly
What is required for disassembly?
-Ribosomal recycling factor (RRF), EF-G-GTP and IF-3 are required for release of uncharged tRNAs from the P-site, & dissociation of the 2 ribosome subunits from mRNA
What are the overall steps of translation in prokaryotes?
Initiation 1. binding of 30S with IFs 2. binding of mRNA and initiator tRNA 3. binding of ribosome 50S & release IFs Elongation 1. binding of new aminoacyl-tRNA at A site 2. formation of new peptide bond 3. translocation Termination 1. binding of RFs 2. hydrolysis of peptidyl-tRNA Disassembly 1. binding of RRF
What is the average mRNA half-life?
E.Coli: 4 min
Humans: 10 hrs
What are the energy requirements for translation?
AA activation : 1ATP*
Assembly of 40S and 60S: 1GTP
Binding of aminoacyl-tRNA: 1GTP (EF-Tu)*
Translocation: 1GTP (EF-G)*
Termination: 1GTP (RF-3)
====> 1 ATP + 2GTP / cycle (4 high E-bonds)
What is protein synthesis different in eukaryotes compared to prokaryotes?
1) In Eukaryotes, protein synthesis occurs in cytoplasm, & transcription/RNA processing in the nucleus.
2) In prokaryotes, it all occurs in the cytoplasm, and processes are coupled.
3) Eukaryotes have more elongation factors than prokaryotes, and only 1 termination factor
4) mRNA has a poly-A tail and 5’-cap
5) mRNA is circularized during the process
6) Regulation of eIF2 functions through reversible phosphorylation
7) 12 initiation factors
What is special about eukaryotic mRNA?
- Methyl guanosine cap at 5’ end (recognition feature for ribosome binding)
- Coding and non-coding sequences
- Poly-A tail at 3’ end
What are nonsense, missense, and silent mutations?
- Nonsense mutation: leads to a termination codon
- Missense mutation: leads to a different aa
- Silent mutation: leads to the same aa
What happens in Huntington’s disease?
- CAG tandem repeats in large numbers
- mRNA is translated into huntingtin protein with abnormal glutamine repeats
- Aggregated proteins cause damage
