Translation in Translation in Prokaryote & Eukaryotes Prokaryote & Eukaryotes Siripan Limsirichaikul Siripan Limsirichaikul
Translation in Translation in
Prokaryote & EukaryotesProkaryote & Eukaryotes
Siripan LimsirichaikulSiripan Limsirichaikul
TranslationTranslation
1. mRNA : RBS, start1. mRNA : RBS, start--stop codonstop codon
2. Ribosome : 2. Ribosome : rRNArRNA + protein+ protein
3. 3. tRNAtRNA : : tRNAtRNA chargingcharging
4. Amino acid : genetic code4. Amino acid : genetic code
DNA3’
3’
5’
5’RNA of gene A
Transcription start site(TSS) (+1)
(_xx) (+xx)Promoter
terminator
upstream downstream
STOP codon
Transcription Unit or
RNA coding regionor
?Gene?
Transcription terminator site
Regulatory region *Gene*
(+ sense)
(- antisense)
START codon
UTR 3’-UTR
5’ 3’
?Gene?
1. 1. Messenger RNAMessenger RNA : : mRNAmRNA
Open Reading Frame (ORF)
UTR = untranslated region
DNA
mRNA
AUG NNN NNN NNN NNN … UAA5’UTR 3’UTR
Ribosome Binding Site (RBS) in prokaryote : Shine-dalgarno seqin Eukaryotes : 5’CAP (m7G)
Start codon
Stop codon
TranscriptionStart site
Transcriptionterminator site
Prokaryote
5’UTR AAAAA…m7G
Eukaryote
AUG NNN NNN NNN NNN … UAA 3’UTR
mRNA
Mature mRNA
Genetic codeGenetic code : : triplet code/ codontriplet code/ codon
43 = 64 codons for 20 amino acids + 3 Stop codons
mRNA
Met Leu Gly
AntisenseDNA
5’ … AUG CUG GGA … 3’
5’ … TCC CAG CAT … 3’3’ … AGG GUC GUA … 5’
Genetic code is not universal
Open reading frameBegin from start codon & End at stop codon
mRNA AUG GCU UUAUGU AGA UAU5’ 3’
Met Ala LeuCys Arg TyrProtein
mRNA
Protein
UGG CUU UAAGUU GAU AU5’ 3’
Trp Leu StopVal
A
Frame 1
Frame 2
Frame 3mRNA
Protein
GGC UUG AAGUUU AUA U5’ 3’
Gly Leu LysPhe
AU
Iso
Ribosome Binding Site (RBS)Ribosome Binding Site (RBS)
ShineShine--DalgarnoDalgarno (SD) (SD) seqseq–– In In prokaryotesprokaryotes–– Bind to ribosome to Bind to ribosome to initiate translationinitiate translation–– Locate Locate upstream of start codonupstream of start codon (AUG)(AUG)–– Consensus Consensus seqseq is is AGG AGG AGGAGG
Ribosome Binding Site (RBS)Ribosome Binding Site (RBS)In EukaryotesIn Eukaryotes
55’’ cap of mRNAcap of mRNA (7(7--methyl methyl guanosineguanosine cap)cap)
polApolA promote promote recyclingrecycling of ribosomeof ribosome
KozakKozak seqseq : upstream of start (: upstream of start (--3)3)–– NotNot a RBSa RBS but but promote promote translation initiationtranslation initiation
2.2. RibosomeRibosome : : rRNArRNA + protein+ protein
Small subunitSmall subunit
–– mRNA binding sitemRNA binding site
–– Decode mRNADecode mRNA
Large subunit : Large subunit : peptidylpeptidyl transferasetransferase
–– E siteE site : Exit: Exit
–– P siteP site : : PeptidylPeptidyl tRNAtRNA binding sitebinding site
–– A siteA site : acceptor site: acceptor site
ProkayotesProkayotes / / EukaryotesEukaryotes
CyclingCycling of Ribosome in cellof Ribosome in cell
3. 3. tRNAtRNA (transfer RNA)(transfer RNA)3’ End : Acceptor arm
Anticodon Loop
tRNAtRNA Charging: Charging: amino acid activation amino acid activation
tRNAtRNA + amino acid = + amino acid = aminoacylaminoacyl tRNAtRNA
STEP 1 : Adenyl-lylation of amino acid
STEP 2 : Adenyl-lylated amino acid +tRNA
tRNAtRNA chargingcharging : step 2 : step 2
tRNAtRNA synthetasesynthetase : : tryptophantryptophansynthetasesynthetase
Specific Specific tRNAtRNAcorresponding corresponding amino acidamino acidATPATP
translationtranslation
InitiationInitiation
ElongationElongation
TerminationTermination
Translation Initiation inTranslation Initiation inEukaryotes Eukaryotes / / ProkaryotesProkaryotes
30s ribosome bind to RBS 30s ribosome bind to RBS (ribosome binding site)(ribosome binding site)
–– 55’’ CAP CAP (Eukaryotes)(Eukaryotes)
–– SD SD (Prokaryotes)(Prokaryotes)
Add first amino acid at start codon (ATG)Add first amino acid at start codon (ATG)
–– Met Met (Eukaryotes)(Eukaryotes)
–– ff--Met Met (Prokaryotes)(Prokaryotes)
TranslationTranslation initiationinitiation in in prokaryoteprokaryote
Initiation factorInitiation factor & small & small
subunit (subunit (16S 16S rRNArRNA) bind ) bind to to SD SD seqseq
AminoacylAminoacyl tRNAtRNA bind to bind to start codonstart codon (AUG)(AUG)
Large subunitLarge subunit joins joins complexcomplex
Small subunit of Ribosome (16s)Small subunit of Ribosome (16s)
bind to mRNA (SD bind to mRNA (SD seqseq))
16s rRNA : 5’ CUCCU 3’
Shine Dalgarno Seq : 5’ AGGAG 3’
Translation Translation initiationinitiation in in prokaryoteprokaryote
TranslationTranslation initiationinitiation in in EukaryoteEukaryote1. 1. PreinitiationPreinitiation complex :complex :
InitationInitation factorfactor + + MetMet--tRNAtRNA + + small subunitsmall subunit (ternary complex)(ternary complex)
2. Initiation complex :2. Initiation complex :
add add capcap--binding complexbinding complex for for binding to 5binding to 5’’ capcap
3. Start codon recognition3. Start codon recognition
ternary complex ternary complex bind to AUGbind to AUG
4. Large subunit (60s) joins 4. Large subunit (60s) joins complexcomplex
Translation Translation elongationelongation
Direction : Direction :
–– 55’’ 33’’ mRNA mRNA
–– N N C terminal end of proteinC terminal end of protein
Peptide bond formationPeptide bond formation
–– ElongationFactor1ElongationFactor1αα--GTPs GTPs –– aminoacylaminoacyltRNAtRNA
–– PeptidylPeptidyl tranferasetranferase in large subunitin large subunit
1. Second aminocyl tRNA bind to A site
3. Peptide bond formation : peptidyl transferase
4. Ribosome translocationtRNA at P E and ejecttRNA at A P
2. GTP hydrolysis …ribosome changes conformation.
Translation Translation elongationelongation
GTPGTP
Translation Translation TerminationTermination
Releasing factorReleasing factor
–– at Aat A--site at STOP codonsite at STOP codon
–– nono tRNAtRNA code stop codoncode stop codon
Stop codonStop codon–– OcherOcher : UAA: UAA–– AmberAmber : UAG: UAG–– Opal Opal : UGA: UGA
Circular Circular PolysomesPolysomes andandRecycling ribosomeRecycling ribosome
PostPost translational translational modificationsmodifications
Cleavage of initiator Cleavage of initiator methioninemethionine–– ff--met in prokaryotesmet in prokaryotes
Disulfide bond formation/ foldingDisulfide bond formation/ folding
Chemical modificationChemical modification–– GlycosylationGlycosylation
–– AcetylationAcetylation
–– PhosphorylationPhosphorylation
Note: for Host selection in genetic engineering, and modified for functional protein