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KAROLINSKA INSTITUTETDepartment of Cell and Molecular Biology

Medical Nobel Institute

Piergiorgio Percipalle, PhD

Tutorial IITutorial IIGene expression:Gene expression:

mRNA translation and protein mRNA translation and protein synthesissynthesis Control of gene transcription and RNA

processing

mRNA translation and protein synthesis

ProgramProgram

mRNA translation

Highlights

• What is mRNA translation?• Molecular mechanisms• Components• Medical relevance• Regulation

mRNA functions as the intermediatebetween DNA and protein

How does the information storedin a linear sequence of nucleotidesin the mRNA translate to a linearsequence of amino acids to obtainproteins?

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Transcription

Protein synthesis

Same language:1 dNTP ≡ 1 rNTP

Different languages:1 rNTP ≠ 1 aa

RNA translation

All cells must be able to translate the nucleotide-based language into proteins

The translation can not be done letter by letterbecause there are 4 bases in the DNA/RNA and 20 different amino acids

Specific problems

The genetic code

mRNA is read in codons (nucleotide triplets), t ex ATG

Every codon specifies an amino acid or a ”STOP” inthe translation process

The genetic code is universalThe genetic code is universal

The genetic code is redundant sinceThe genetic code is redundant since many amino many amino acids are specified by several codons.acids are specified by several codons.

Black letters: codons (nucleotide triplets)Red letters: amino acids

The genetic code

How are nucleotides coupled with amino acids?

Adaptor molecules match amino acidsto mRNA codons

The adaptor molecule is called transfer-RNA (tRNA)

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AnticoAnticodondon

Codon binding Codon binding sitesite

Amino acid Amino acid binding sitebinding site

33’’ endend

The 3D structure of tRNA is L-shapedand exhibits binding sites for both

amino acids and codons

Black letters: codons (nucleotide triplets)Red letters: amino acids

The genetic code

The The ””WobbleWobble”” effect ineffect in base pairingbase pairingbetween codons andbetween codons and anticodonsanticodons

A specific enzyme loadstRNA molecules with an amino acid

The enzyme is called amino acyl t-RNA synthetase

1)2)

The genetic code is translatedwith the help of two different adaptors

Net result: one amino acid is chosen by one codon

Amino acyl tRNA

The new amino acids are incorporatedin the growing polypeptide chain

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Where does translation take place?

Ribosomes

Eukaryoticribosome

Prokaryoticribosome

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RibosomesRibosomes are very complex catalytic machineriescontaining more than 50 different proteins as well as ribosomal RNA (rRNA)

Ribosomes are very conserved between prokaryotic and eukaryotic organisms

Active ribosomes conist of a large and a small subunit

In eukaryotes, active ribosomes are only present inthe cytoplasm

Antibiotics and mRNA translation

Antibiotics are designed against processes and structure which are unique for microbes and are not found in eukaryotes

There are antibioticsThere are antibiotics (eg.(eg. kanamycinkanamycin) ) which which specifically block bacterial ribosomesspecifically block bacterial ribosomes

There are slight differences between eukaryotic and prokaryotic ribosomes and these differences are targeted by antibiotics

Protein synthesis is a dynamicprocess

How can the ribosome facilitateand coordinate all the molecularmechanisms that take place for efficient mRNA translation?

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Ribosomes contain four binding sites

mRNA binding site

tRNA binding sites

There are three main stepsin mRNA translation

Binding of amino acyl-tRNA to A-site

The C-terminus end of the peptide in theP-site is coupled to the N-terminus ofamino acid in the A-site

Conformational changesConformational changes cause the shift cause the shift of the mRNAof the mRNA by exactlyby exactly threethree nucleotidesnucleotidessoso thatthat a new Aa new A--sitesite becomes availablebecomes available

Translation of an mRNA molecule…..

Peptidyl transferase

Conformational changes in the mRNA

….require the help of elongation factors

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Initiation and termination of translationrequires specific signals

Initiation requires a specific AUG codon, which exclusively binds to a tRNA carrying a methionine

Translation is terminated when stop codons (UAA, UAG, UGA) are encountered by the ribosome

None of the tRNA molecules recognizes stop codons

Initiation requires initiation factorsInitiator tRNA binds the small ribosomal subunit

The small ribosomal subunit recognizes the 5’-cap structure in the mRNA (it also needs initiation factors eIF4E andeIF4G)

The small ribosomal subunit scans the mRNA until it reaches the AUG codon

The large ribosomal subunit binds the small ribosomal subunit to reconstitute an active ribosome. The initiation factors are dissociated

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Initiation of translation

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Termination of translation

The ribosomal subunits dissociate

The proteins which areready leave ribosomes

Proteins are made in polyribosomes

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The synthesis of most proteins takes between 20 seconds and few minutes

Quality control of newly synthesized proteins ?

Proteins are folded co-translationallyProteins acquire their secondary structure during the translation process

Several mechanisms garanteecorrect protein folding

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Abnormally folded proteins can aggregate and give rise to disease

Prion, Huntington and Alzheimer are the result of accumulation of wrongly folded proteins which buildfilaments that can not be degraded and impair cellular function

A long way from DNA to protein