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)