translation - ulisboabmg.fc.ul.pt/disciplinas/fundbiolmolec/16translationaula.pdftranslation in...
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Translation
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Concept of colinearity: a continuous sequence of nucleotides in DNAencodes a continuous sequence of amino acids in a protein
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Para além do fenómeno do wobble,…
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… há que considerar
• Desvios ao código genético– Excepções ao código genético universal (constituitivos)- desvios
muito observados em genomas mitocondriais– Pontuais (site-specific variations)- geralmente envolvem o codão
stop. • Ex. inserção da selenocisteína no codão UGA
• Ambiguidades no código genético– Codão de iniciação: AUG, GUG, UUG, CUG– fMet-tRNAfMet
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Incorporation of selenocysteine into a growing polypeptide chain
A specialized tRNA is charged with serine by the normal seryl-tRNA synthetase, and the serine is subsequently converted enzymatically to selenocysteine
A specific RNA structure in the mRNA (a stem and loop structure with a particular nucleotidesequence) signals that selenocysteine is to be inserted at the neighboring UGA codon. This event requires the participation of a selenocysteine-specific translation factor
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Protein Organism
Prokaryotic enzymesFormatedehydrogenase
Clostridium thermoaceticum, Clostridium thermoautotrophicum, Enterobacteraerogenes, Escherichia coli, Methanococcus vaniellii
Glycine reductase Clostridium purinolyticum, Clostridium sticklandii
NiFeSe hydrogenase Desulfomicrobium baculatum, Methanococcus voltae
Eukaryotic enzymes
Glutathione peroxidase
Human, cow, rat, mouse
Selenoprotein P Human, cow, rat
Selenoprotein W Rat
Type 1 deiodinase Human, rat, mouse, dog
Type 2 deiodinase Frog
Type 3 deiodinase Human, rat, frog
Proteins containing selenocystein
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Unusual types of aminoacylation
Genomes 11.5
Selenocysteine is the same as cysteine but with the sulfur replaced with a selenium atom in the R group
The special tRNA used in initiation of translation in bacteria is aminoacylatedwith methionine, which is then converted to N-formylmethionine (transformilase)
In some bacteria, tRNAGln is aminoacylated with glutamicacid, which is then converted to glutamine by transamidation
tRNASeCys in various organisms isinitially aminoacylated with serine
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Translation in prokaryotes
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Procaryotic ribosomes initiate transcription at ribosome-binding sites
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Structure of a typical bacterial mRNA molecule
Shine-Dalgarno sequences can be located anywhere (but specifically) along an mRNA molecule.
This permits bacteria to synthesize more than one type of protein from a single mRNA molecule
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Shine-Dalgarno consensus sequencevs
Ribosome binding site
*
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rRNArRNA 16S 16S bacteriannobacterianno
Emparelhamento de bases que confere estrutura a rRNA 16S
Posições dentro do rRNA 16S de E. coli que interagem com a proteína ribossomal 5S
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Prokaryotic ribosome(functional sites)
PeptidylTransferase(rRNA 23S)
3’ end 16S rRNA
f-Met enters at the P site
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In prokaryotic cells, transcription and translation take place simultaneously
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An mRNA molecule may be transcribed simultaneouslyby several ribosomes
The mRNA is translated in the 5 -to-3 direction, and the N-terminal end of a protein is made first, with each cycle adding one amino acid to the C-terminus of the polypeptide chain
Ribossomesorganized in polissomes
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Four steps involved in translation
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Dynamicequilibrium
IF3 binds to the small unit of ribosomepreventing large subunit from binding
INITIATION of translation in bacterial cells requires severalinitiation factors and GTP
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F-Met-tRNA forms a complexwith IF-2 and GTP. IF-2 directs initiator tRNAMet
EF-1, blocks A site and is responsible for conformational modification of small subunit
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IF-1, IF-2 and IF-3 dissociate fromthe complex, GTP is hydrolyzed to GDP and the large subunit joins to create the 70S initiation complex
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The ELONGATION of translation comprises three steps
Complex EF-Tu, EF-Ts, GTP and charged tRNAEF-Tu, directs the next tRNA
Charged tRNA is placed into theA site, GTP is cleaved andEF-Tu-GDP complex is released
EF-G, mediatestranslocation
The peptide bond formationreleases the aa in the P sitefrom its tRNA
The position at which the growing peptide chain is attached to a tRNA does not change during the elongation cycle: it is always linked to the tRNA present in the P site of the large subunit
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TERMINATION of translation
Peptide release fromthe tRNA in the P site
Translation ends when a stop codon is encountered; there is no tRNA with an anticodonthat can pair with the codon in the site A
RF-1 UAA UAGRF-2 UAA UGARF-3 stimulates dissociation of RF-1 and RF-2
RRF- ribosome recycling factor
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Translation in eukaryotes
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Translation in eukaryotes
• Efficient translation initiation also requires the polypoly--A tailA tail of the mRNA bound by poly-A-binding proteins which, in turn, interact with eIF4G. In this way, the translation apparatus ascertains that both ends of the mRNA are intact before initiating translation
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An eukaryotic polyribosomeSchematic drawing showing how a series of ribosomes can simultaneously translate the same eucaryotic mRNA molecule
Electron micrograph of a polyribosomefrom a eucaryotic cell
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The competition between mRNA translation and mRNA decay
The same two features of mRNA the 5’ cap and the 3’ poly-A site are used in both translation initiation and deadenylation-dependent mRNA decay
The enzyme (called DAN) that shortens the poly-A tail in the 3’ to 5’ direction associates with the 5’ cap
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Two mechanisms of translation initiation
The cap-dependent mechanism requires a set of initiation factors whose assembly on the mRNA is stimulated by the presence of a 5’ cap and a poly-A tail
The IRES-dependent mechanism requires only a subset of the normal translation initiating factors, and these assemble directly on the folded IRES
Internal ribosome entry sites
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The initiation phase of protein synthesis in eucaryotes
eIF2 binds to tRNAMet
eIF4E
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eIF4A and eIF4Bhave helicase activity
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eEF-2, translocationfactor, similar to EF-G
eEF-1, elongationfactor, similar to EF-Tu
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eRF-1 similar to tRNA andrecognizes termination codon
eRF-3 similar to bacteria RF-3
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Regulation of gene expression attranslational level
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– Translation initiation efficiency (includes RBS affinity in prokaryotes)
– Polarity (in prokaryotes)
– Codon usage (codon preference or codon bias)
– mRNA degradation
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Production of distinct amylase mRNA molecules by differentsplicing events in cells of the salivary gland and liver of the
mouse affects the translation efficiency and though the level ofamylase synthesis
Transcripts with different 5’-UTR
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Negative translational control
• This form of control is mediated by a sequence-specific RNA-binding protein that acts as a translation repressor. Binding of the protein to an mRNA molecule decreases the translation of the mRNA
• The illustration is modeled on the mechanism that causes more ferritin(an iron storage protein) to be synthesized when the free iron concentration in the cytosol rises; the iron-sensitive translation repressor protein is called aconitase
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Two posttranscriptional controls mediated by iron
In response to an increase in ironconcentration in the cytosol, a cell increases its synthesis of ferritinin order to bind the extra iron…
… and decreases synthesis of transferrin receptors in order toimport less iron across the plasma membrane
Both responses are mediated by the same iron-responsive regulatory protein, aconitase, which recognizes common features in a stem-and-loop structure in the mRNAs encoding ferritin and transferrin receptor
Transferrin receptor and ferritin are regulated by different types of mechanisms, their levels respond oppositely to iron concentrationseven though they are regulated by the sameiron-responsive regulatory protein
IRE- iron response element
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Steps at which eucaryotic gene expression can becontrolled
1- transcriptional activatorsmethylationchromatin remodelation…
2- altenative splicingRNA editingRNAi…
4- polyadenylation/deanylation5’-UTR binding proteinsRNAi…