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