Conceptofcolinearity: a continuous sequence of nucleotides in DNA encodes a continuous sequence of amino acids in a protein

Transcription

1 Translation

2 Conceptofcolinearity: a continuous sequence of nucleotides in DNA encodes a continuous sequence of amino acids in a protein

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4 Para além do fenómeno do wobble,

5 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-trna fmet

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7 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 nucleotide sequence) signals that selenocysteine is to be inserted at the neighboring UGA codon. This event requires the participation of a selenocysteine-specific translation factor

8 Proteins containing selenocystein Protein Organism Prokaryotic enzymes Formate dehydrogenase Glycine reductase NiFeSe hydrogenase Clostridium thermoaceticum, Clostridium thermoautotrophicum, Enterobacter aerogenes, Escherichia coli, Methanococcus vaniellii Clostridium purinolyticum, Clostridium sticklandii Desulfomicrobium baculatum, Methanococcus voltae Eukaryotic enzymes Glutathione peroxidase Selenoprotein P Selenoprotein W Type 1 deiodinase Type 2 deiodinase Type 3 deiodinase Human, cow, rat, mouse Human, cow, rat Rat Human, rat, mouse, dog Frog Human, rat, frog

9 Unusual types of aminoacylation In some bacteria, trnagln is aminoacylated with glutamic acid, which is then converted to glutamine by transamidation The special trna used in initiation of translation in bacteria is aminoacylated with methionine, which is then converted to N-formylmethionine (transformilase) trnasecys in various organisms is initially aminoacylated with serine Selenocysteine is the same as cysteine but with the sulfur replaced with a selenium atom in the R group Genomes 11.5

10 Translation in prokaryotes

11 Procaryotic ribosomes initiate transcription at ribosome-binding sites

12 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

13 Shine-Dalgarno consensus sequence vs Ribosome binding site *

14 rrna 16S bacterianno Emparelhamento de bases que confere estrutura a rrna 16S Posições dentro do rrna 16S de E. coli que interagem com a proteína ribossomal 5S

15 Prokaryotic ribosome (functional sites) Peptidyl Transferase (rrna 23S) 3 end 16S rrna f-metentersatthep site

16 In prokaryotic cells, transcription and translation take place simultaneously

17 An mrna molecule may be transcribed simultaneously by several ribosomes Ribossomes organized in polissomes 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

18 Four steps involved in translation

19 INITIATION of translation in bacterial cells requires several initiation factors and GTP Dynamic equilibrium IF3 binds to the small unit of ribosome preventing large subunit from binding

20 F-Met-tRNA forms a complex with IF-2 and GTP. IF-2 directs initiator trna Met EF-1, blocks A site and is responsible for conformational modification of small subunit

21 IF-1, IF-2 and IF-3 dissociate from the complex, GTP is hydrolyzed to GDP and the large subunit joins to create the 70S initiation complex

22 The ELONGATION of translation comprises three steps Charged trna is placed into the A site, GTP is cleaved and EF-Tu-GDP complex is released Complex EF-Tu, EF-Ts, GTP and charged trna EF-Tu, directs the next trna The peptide bond formation releasestheaainthep site from its trna EF-G, mediates translocation 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

23 TERMINATION of translation Translation ends when a stop codon is encountered; there is no trna with an anticodon that can pair with the codon in the site A Peptide release from the trna in the P site RF-1 RF-2 RF-3 UAA UAG UAA UGA stimulates dissociation of RF-1 and RF-2 RRF- ribosome recycling factor

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25 Translation in eukaryotes

26 Translation in eukaryotes Efficient translation initiation also requires the poly-a A tail of the mrna bound by poly-abinding 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

27 An eukaryotic polyribosome Schematic drawing showing how a series of ribosomes can simultaneously translate the same eucaryotic mrna molecule Electron micrograph of a polyribosome from a eucaryotic cell

28 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

29 Two mechanisms of translation initiation Internal ribosome entry sites 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

30 The initiation phase of protein synthesis in eucaryotes eif4e eif2 binds to trna Met

31 eif4a and eif4b have helicase activity

32 eef-1, elongation factor, similar to EF-Tu eef-2, translocation factor, similar to EF-G

33 erf-1 similar to trna and recognizes termination codon erf-3 similar to bacteria RF-3

34 Regulation of gene expression at translational level

35 Translation initiation efficiency (includes RBS affinity in prokaryotes) Polarity (in prokaryotes) Codon usage (codon preference or codon bias) mrna degradation

36 Production of distinct amylase mrna molecules by different splicing events in cells of the salivary gland and liver of the mouse affects the translation efficiency and though the level of amylase synthesis Transcripts with different 5 -UTR

37 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

38 Two posttranscriptional controls mediated by iron 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 IRE- iron response element Transferrin receptor and ferritin are regulated by different types of mechanisms, their levels respond oppositely to iron concentrations even though they are regulated by the same iron-responsive regulatory protein In response to an increase in iron concentration in the cytosol, a cell increases its synthesis of ferritin in order to bind the extra iron and decreases synthesis of transferrin receptors in order to import less iron across the plasma membrane

39 Steps at which eucaryotic gene expression can be controlled 1- transcriptional activators methylation chromatin remodelation 2- altenative splicing RNA editing RNAi 4- polyadenylation/deanylation 5 -UTR binding proteins RNAi