Translation - Állatorvostudományi Egyetem lessons 27-28... · Initiation of translation in...
Transcript of Translation - Állatorvostudományi Egyetem lessons 27-28... · Initiation of translation in...
Translation
Translation
• Translation = Biosynthesis of proteins based on mRNA � expression of genetic information
• For translation the followings are needed:• mRNA: carries the genetic information• tRNA: transports activated amino acids for
translation• Ribosome: subcellular organ where translation
takes place• Numerous protein factors
Structure of tRNA (transfer RNA)
• Intramolecular base pairing �double stranded sections
• Three loops (~shamrock shape)• DHU-loop: binds aminoacyl-tRNA-
synthetase• Contains dihydro-uridin-monophosphate
(no double bond in uracil)
• Anticodon-loop: binds codons of mRNA
• TφC-loop: binds large subunit of ribosome
• T = thymidine-monophosphate (thymine in RNA!)
• Φ = pseudouridine-monophosphate (uracil binds to ribose with C atom 5)
DHU-loop
Anticodon-
loop
TφC-loop
3’-end: CCA-sequence � binds
carboxylgroup of transported amino
acid with ester bond
Structure of tRNA(transfer RNA)
http://en.wikipedia.org/wiki/Shamrock
http://classes.midlandstech.edu/carterp/Courses/bio101/chap14/chap14.htm
Activation of amino acids
Binding of adequate amino acid with the 3’-CCA-sequence of tRNA:
Aminoacyl-tRNA-synthetase: specific for the tRNA and forthe amino acid
• Aminoacyl-tRNA-synthetase binds ATP
• ATP+Amino acid � aminoacyl-AMP + PpinAminoacyl-AMP: the amino acid binds to the phosphate group of AMP with acid anhydrid bond
• Aminoacyl-tRNA-synthetase binds to the DHU loop of tRNA
• Aminoacyl-AMP + tRNA � aminoacyl-tRNA + AMPAminoacyl-tRNA: the amino acid binds to the 3-OH-group of theribose of AMP at CCA-sequence of 3’-end of tRNA
Structure of ribosomes
• Ribosome is composed of ribosomal RNA molecules (rRNA) and proteins
• Different types of rRNA can be characterisedaccording to their sedimentationcoefficients (unit is Svedberg = S)
• Large subunit:• Eukaryotes: 5S rRNA; 5,8S rRNA; 28S rRNA + 49
protein
• Prokaryotes: 5S rRNA; 23S rRNA + 34 protein
• Small subunit:• Eukaryotes: 18S rRNA +33 protein
• Prokaryotes: 16S rRNA + 21 protein
Structure of ribosomes
Binding sites of ribosome
Binding sites of large
ribosomal subunit:
A = Aminoacyl-tRNA
P = Peptidyl-tRNA
E = Exit
Binding site of small
ribosomal subunit:
mRNA-binding site
Translation in eukaryotes
Place
• Cytoplasma(mRNA have to transport out from the nucleus)
• Rough endoplasmatic reticulum:membran with ribosomes
Initiation of translation ineukaryotes
1. Small ribosomal subunit + eIF-2-GTP + other eIF-s + tRNAMet
� Prae-initiation complex
2. Kozak-scanning mechanism: the prae-initiation complex rolls along the mRNA searching for the start codon: AUG = methionine-codon (energy consumption: 1 ATP/nucleotide)
3. Recognised start codon � small ribosomal subunit (18S rRNS) binds to the Kozak-sequence of mRNA
4. GTP bound to eIF-2 hydrolyses to GDP + Pin � large ribosomal subunit binds to the small one and all the eIF-s dissociate
� Initiation complex
eIF = eukaryontischer Initiationsfaktor
tRNAMet = Methionyl-tRNA (initiator tRNA)
Initiation of translation in eukaryotes
Elongation of translation in eukaryotes
• At the end of initiation:• Initiation complex � methionyl-tRNA binds to the P-
site of large subunit with its TφC-loop
• A-site of large ribosomal subunit is free
• Elongation consists of cycles:
• All cycles have three steps
• Peptide chain gets longer by one amino acid in each cycle
Elongation of translation in eukaryotes
1. Binding of aminoacyl-tRNA to A-site of large ribosomal subunit
• EF-1α + GTP complex is needed for binding (energy consumption: GTP hydrolyses to GDP + Pin) (EF = elongation factor)
• Aminoacyl-tRNA binds to the A-site of large ribosomal subunit with its TφC-loop and to the next codon of mRNA with its anticodon-loop (only that aminoacyl-tRNA can bind of wich anticodon-loop is complementary to the codon of mRNA)
Elongation of translation in eukaryotes
2. Methionin (or from the 2nd cycle: the peptide chain) is placed from the methionyl-tRNA (or from the 2nd cycle: peptidyl-tRNA) onto the newly bound aminoacyl-tRNA (P�A): peptidyl-transferase
• Peptidyl-transferase is a ribozym: this is the 28S rRNA of large ribosomal subunit
• Peptidyl-transferase needs NO energy for this step
• By this step, a new peptide (amide) bond is formed betweeen methionine (or from the 2nd cycle: the peptide chain) and the newly bound amino acid (aminoacyl-tRNA) � dipeptidyl-tRNA (or from the 2nd cycle: longer peptidyl-tRNA)
• The „empty” tRNA goes to E-site from P-site � then it dissociates
Elongation of translation in eukaryotes
3. Translocation: dipeptidyl-tRNA (or from the 2nd cycle: peptidyl-tRNA) is placed onto P-site from A-site
• Translocation is catalysed by translocase
• Translocation needs EF-2 + GTP complex (energy consumption: GTP hydrolyses to GDP + Pin)
• Ribosome rolls along the mRNA by three nucleotides (one codon)
Elongation of translation in eukaryotes
• At the end of elongation:• Dipeptidyl-tRNA (or from the 2nd cycle: peptidyl-tRNA)
binds to the P-site of large ribosomal subunit with its TφC-loop
• A-site of large ribosomal subunit is free
• In next cycle?:• Binding of new aminoacyl-tRNA to A-site
• Peptidyl-transferase � tripeptidyl-tRNA on A-site
• Translocation � tripeptidyl-tRNA is placed onto P-site from A-site � A-site gets free
Fsimilarly in all cycles
• Peptide chain grows by one amino acid in each cycle
Overview of
elongation
Termination of translation(in pro- and eukaryotes)
• At stop codon (UAA, UAG or UGA) no aminoacyl-tRNA can bind to A-site � PRF protein binds to A-site instead
• PRF = protein releasing factor
• PRF splits peptide chain of peptidyl-tRNA (GTP-consumption!) � the newly synthesized protein chain gets free
• Two ribosomal subunits and mRNA dissociate
Termination of translation(in pro- and eukaryotes)
• Deliberation of the newly synthesized protein chain and dissociation of ribosomal subunits caused by PRF
Initiation of translation in prokaryotes
1. Small ribosomal subunit + IF-1 and IF-3
2. + IF-2-GTP + tRNAini + mRNA
� 30S initiation complex
No Kozak-scanning, small ribosomal subunit (16S rRNA) binds to the Shine-Dalgarno-sequence (RBS = ribosome binding site) of mRNA
Initiation of translation in prokaryotes
3. IF-1 and IF-3 dissociate, GTP hydrolyses �large ribosomal subunit binds
� 70S initiation complex
Major differences compared to eukaryotes:
•3 IF-s are needed only ↔ eukaryotes need more
•Initiator (first) amino acid: N-formyl-methionine
•No Kozak-scanning mechanism
Initiation of translation in prokaryotes
Elongation of translation in prokaryotes
• Different elongation factors: • EF-1α � EF-Tu
• EF-2 � EF-G
• Transcription and translation happen simultaneously
• One mRNA binds to more than one ribosomes � Translation happen parallelly from all gene transcripts of mRNA (polycistronic!) �polyribosome
Elongation of translation in prokaryotes
• Polycistronic mRNA � polyribosome
https://www.youtube.com/watch?v=Jml8CFBWcDs
Energy balance of translation
• Initiation: 1 GTP (in complex with eIF-2 or IF-2)
• Kozak-scanning mechanism (eukaryotes only):1 ATP/nucleotide
• Elongation: 4 ATP/amino acid• For the activation of amino acid: 2 ATP
(ATP →AMP+PPin, for the resynthesis the energy of 2 ATPs are needed)
• Binding of aminoacyl-tRNA to A-site:1 GTP (in complex with EF-1α or EF-Tu)
• Translocation: 1 GTP (in complex with EF-2 or EF-G)
• Termination: 1 GTP (for PRF)