BIO353 – lecture 11: translation

Langenscheidt
Standard Dictionary

Protein
DNA - RNA
RNA - AA

shf – 2021
Translation: general overview - central dogma

RNA synthesis TRANSCRIPTION

protein synthesis TRANSLATION

shf – 2021
 Translation: basic ingredients

processed mRNA

ribosome

protein synthesis

20 amino acids charged tRNAs

shf – 2021
 Translation: general overview

DNA: a 4 character code Protein: 20 building blocks

• one letter word 4 possibilities

• two letter word 16 possibilities < than needed

• three letter word 64 possibilities > than needed

degeneration of the genetic code

shf – 2021
 Translation: general overview

degeneration of the genetic code

alternative start codons in prokaryotes

shf – 2021
 Translation: general overview

AUG AAA GCA AUU UUC GUA CUG AAA GGU UGG UGG UGA
Frame 1
M K A I F V L K G W W stop

A UGA AAG CAA UUU UCG UAC UGA AAG GUU GGU GGU GA
Frame 2
stop K Q F S Y stop K V G G

AU GAA AGC AAU UUU CGU ACU GAA AGG UUG GUG GUG A
Frame 3
E S N F R T E R L V V

shf – 2021
 Translation: initiation - prokaryotes vs eukaryotes

ribosome binding site

polycistronic gene
cistron 1 cistron 2 cistron 3
(= ORF 1) (= ORF 2) (= ORF 3)

shf – 2021
Translation: initiation in prokaryotes – Shine-Dalgarno sequence

araB UUUGGAUGGAGUGAAACGAUGGCGAUU
gelE AGCCUAAUGGAGCGAAUUAUGAGAGUU
lacI CAAUUCAGGGUGGUGAUUGUGAAACCA
lacZ UUCACACAGGAAACAGCUAUGACCAUG
trpE CAAAAUUAGAGAAUAACAAUGCAAACA
trpL GUAAAAAGGGUAUCGACAAUGAAAGCA
talA UUUCAAGGAGUAUUUCCUAUGAACGAG
dsdX CACAGGGGAAGGUGAGAUAUGCACUCU

Shine-Dalgarno Start codon

sequence
= ribosome binding site

shf – 2021
 Translation: initiation in prokaryotes – Shine-Dalgarno sequence

araB UUUGGAUGGAGUGAAACGAUGGCGAUU
gelE AGCCUAAUGGAGCGAAUUAUGAGAGUU
lacI CAAUUCAGGGUGGUGAUUGUGAAACCA
lacZ UUCACACAGGAAACAGCUAUGACCAUG
trpE CAAAAUUAGAGAAUAACAAUGCAAACA
trpL GUAAAAAGGGUAUCGACAAUGAAAGCA
talA UUUCAAGGAGUAUUUCCUAUGAACGAG
dsdX CACAGGGGAAGGUGAGAUAUGCACUCU

3’-end of 16S RNA AUUCCUCCACUAG

shf – 2021
 Translation: initiation in prokaryotes – Shine-Dalgarno sequence

ribosomal RNA

Shine-Dalgarno sequence

polycistronic gene

shf – 2021
 Translation: initiation - prokaryotes vs eukaryotes

ribosomal RNA

Shine-Dalgarno sequence

polycistronic gene

Kozak sequence

monocistronic gene
shf – 2021
 Translation: tRNA structure and function

sequence added post-transcriptionally

(CCA-adding enzyme)

amino acid

tRNA (conceptual structure) tRNA (folded structure)

shf – 2021
Translation: tRNA structure and function

à structural stability (Y)

à anticodon ambiguity (inosine)

à no degradation (dihydrouridine)

shf – 2021
Translation: and the genetic code(s)

genetic code second genetic code

RNA à amino acid amino acid à tRNA

shf – 2021
Translation: charging (= loading) tRNAs with amino acids

charging of tRNA with AA

shf – 2021
Translation: charging tRNAs with amino acids - step 1

adenylylation

shf – 2021
Translation: charging tRNAs with amino acids - step 2

tRNA charging

charged tRNA

shf – 2021
Translation: charging tRNAs with amino acids - tRNA synthetases

AA à 3’-OH AA à 2’-OH

shf – 2021
Translation: charging tRNAs with amino acids - tRNA synthetases

degeneration of the genetic code

one tRNA synthetase per AA but multiple tRNAs for each AA

shf – 2021
 Translation: charging tRNAs with amino acids - tRNA synthetases

Aminoacyl-tRNA
synthetase

amino acid

ATP

tRNA

AMP + 2 Pi

charged
tRNA

shf – 2021
 Translation: charging tRNAs with amino acids - tRNA synthetases

discriminator base
Aminoacyl-tRNA tRNA
synthetase

amino acid

ATP
amino acid

tRNA

AMP + 2 Pi

charged anticodon
tRNA

tRNA synthetase

shf – 2021
Translation: charging tRNAs with amino acids - tRNA synthetases

shf – 2021
Translation: charging tRNAs with amino acids - tRNA synthetases

easy not so easy

à exclusion in editing pocket

shf – 2021
Translation: the structure of the ribosome

Theodor Svedberg
(1884 – 1971)

shf – 2021
 Translation: the structure of the ribosome

small subunit à 21 proteins large subunit à 34 proteins

Kaltschmidt, E. and H.G. Wittmann, Ribosomal proteins XII: Number of proteins in small and large ribosomal subunits of Escherichia coli as
determined by two-dimensional gel electrophoresis. PNAS USA, (1970, 67:1277–78.)

shf – 2021
Translation: the structure of the ribosome

prokaryotes

16S rRNA

shf – 2021
Translation: the structure of the ribosome

prokaryotes

peptidyl-transferase center

decoding center

shf – 2021
Translation: the structure of the ribosome

eukaryotes prokaryotes

shf – 2021
Translation: the ribosome cycle - prokaryotes

shf – 2021
 Translation: polyribosomes (polysomes)

prokaryotes cotranscriptional

in cytoplasm
eukaryotes

30 nt

~80 nt
shf – 2021
Translation: polypeptide synthesis reaction

amino acid

amino acid

peptidyl transferase
reaction

shf – 2021
Translation: the ribosome structure - EPA sites

shf – 2021
Translation: the ribosome structure - EPA sites

large subunit small subunit

decoding center

peptidyl transferase
mostly rRNA
center
devoid of protein

shf – 2021
 Translation: the ribosome structure - EPA sites

P A
P
E A

AA AA

kink in template RNA proximity of acceptor stem

of P and A site tRNAs

shf – 2021
 Translation: the ribosome structure - EPA sites

exit tunnel (polypeptide)

large subunit

A
P
E
shf – 2021
Translation: initiation in prokaryotes

identification of
initiation translation start codon

elongation polypeptide synthesis

termination termination of chain elongation

clearing of ribosome

shf – 2021
Translation: initiation in prokaryotes

step 1 step 2

binding of binding of
mRNA and initiator tRNA large ribosomal subunit
(any order)

shf – 2021
Translation: initiation in prokaryotes - start site

shf – 2021
Translation: initiation in prokaryotes - start site

initiator tRNA

vs

shf – 2021
Translation: initiation in prokaryotes - start site

fMet resembles a peptide while occupying the P site

peptidyl-tRNA fMet-tRNAifMet Met-tRNAMet

shf – 2021
Translation: initiation in prokaryotes - regulatory factors

dissociation of
the small subunit

occupation of
E site

shf – 2021
Translation: initiation in prokaryotes - regulatory factors

occupation of
A site

dissociation of
the small subunit initiator tRNA
loading to P site

occupation of
E site

shf – 2021
Translation: initiation in prokaryotes - regulatory factors

binding of
large subunit

factor binding
release of IF3 center

release of IF1 / 2
(GTP hydrolysis by IF2)

shf – 2021
Translation: initiation in eukaryotes - regulatory factors

ribosome

dissociation of
the small subunit

occupation of
E and A site

Met-tRNAMet
loading to P site

43S preinitiation
complex
shf – 2021
Translation: initiation in eukaryotes - regulatory factors and events

ribosome mRNA

recognition of 5’-CAP

binding of mRNA to 43S PIC

helicase activity /
removal of secondary structure

43S preinitiation
complex
shf – 2021
Translation: initiation in eukaryotes - regulatory factors and events

ribosome mRNA

loading of mRNA to 43S PIC

shf – 2021
Translation: translation in eukaryotes - reinitiation

binding / stimulation of EFs

re-initiation

shf – 2021
Translation: initiation in eukaryotes - scanning

scanning à

shf – 2021
Translation: initiation in eukaryotes - subunit recruitment

recruitment of large subunit

shf – 2021
Translation: initiation in eukaryotes - subunit recruitment

eviction of IFs

à elongation

shf – 2021
Translation: initiation in eukaryotes - internal ribosome entry sites (IRES)

shf – 2021
Translation: elongation - steps

shf – 2021
Translation: elongation - steps

shf – 2021
Translation: elongation - steps

shf – 2021
Translation: elongation - proofreading (EF-Tu)

1. - proofreading by 2. - proofreading by
16S rRNA EF-Tu

shf – 2021
Translation: elongation - proofreading (accommodation)

3. - proofreading by
accommodation

shf – 2021
Translation: elongation - translocation

shf – 2021
Translation: elongation - translocation

shf – 2021
Translation: elongation - translocation

EF-Tu / aminoacyl - tRNA EF-G-GDP

shf – 2021
Translation: termination - release factors

t-RNA

release factor

shf – 2021
Translation: termination - release factors

shf – 2021
Translation: termination - release factors

shf – 2021
Translation: termination - ribosome recycling

shf – 2021
Translation: what if something goes wrong? - no STOP codon

shf – 2021
Translation: what if something goes wrong? - no STOP codon

re-initiation in prokaryotes - tmRNA

shf – 2021
Translation: what if something goes wrong? - no STOP codon

re-initiation in prokaryotes - tmRNA

shf – 2021
Translation: what if something goes wrong? - stalling

no go-mediated decay nonstop-mediated decay

shf – 2021
Translation: what if something goes wrong? - no STOP codon

eukaryotes - nonstop-mediated decay

shf – 2021
Translation: what if something goes wrong? - premature STOP codon

nonsense-mediated decay

shf – 2021
Translation: what if something goes wrong? - premature STOP codon

nonsense-mediated decay

shf – 2021