Translation

Concept of colinearity: a continuous sequence of nucleotides in DNAencodes a continuous sequence of amino acids in a protein

Para além do fenómeno do wobble,…

… 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

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

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

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

Translation in prokaryotes

Procaryotic ribosomes initiate transcription at ribosome-binding sites

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

Shine-Dalgarno consensus sequencevs

Ribosome binding site

*

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

Prokaryotic ribosome(functional sites)

PeptidylTransferase(rRNA 23S)

3’ end 16S rRNA

f-Met enters at the P site

In prokaryotic cells, transcription and translation take place simultaneously

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

Four steps involved in translation

Dynamicequilibrium

IF3 binds to the small unit of ribosomepreventing large subunit from binding

INITIATION of translation in bacterial cells requires severalinitiation factors and GTP

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

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

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

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

Translation in eukaryotes

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

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

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

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

The initiation phase of protein synthesis in eucaryotes

eIF2 binds to tRNAMet

eIF4E

eIF4A and eIF4Bhave helicase activity

eEF-2, translocationfactor, similar to EF-G

eEF-1, elongationfactor, similar to EF-Tu

eRF-1 similar to tRNA andrecognizes termination codon

eRF-3 similar to bacteria RF-3

Regulation of gene expression attranslational level

– Translation initiation efficiency (includes RBS affinity in prokaryotes)

– Polarity (in prokaryotes)

– Codon usage (codon preference or codon bias)

– mRNA degradation

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

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

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

Steps at which eucaryotic gene expression can becontrolled

1- transcriptional activatorsmethylationchromatin remodelation…

2- altenative splicingRNA editingRNAi…

4- polyadenylation/deanylation5’-UTR binding proteinsRNAi…