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Translation
Concept of colinearity: a continuous sequence of nucleotides in DNA encodes
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 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
Proteins containing selenocystein
Protein
Organism
Prokaryotic
enzymes
Formate
dehydrogenase
Clostridium thermoaceticum, Clostridium thermoautotrophicum, Enterobacter
aerogenes,
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
Codões de iniciação da tradução: AUG, GUG, UUG, CUG
Met-tRNAMet
fMet-tRNAiMet
3’
3’
5’
3’
A*
UAC
5’
5’
3’
5’
AUG 3’
A
AAC
5’
5’
GUG 3’ A não modificação da adenosina
a 3’ do anticodão do tRNAi permite
uma certa flexibilidade de
emparelhamento do fMet-tRNAiMet
A*- adenosina alquilada
Unusual types of aminoacylation
The special tRNA used in initiation of
translation in bacteria is aminoacylated
with methionine, which is then converted
to N-formylmethionine (transformilase)
Bacteria IF2 only recognizes fMet-tRNAiMet
Eucaryotic eIF2 only recognizes Met-tRNAiMet
Genomes 11.5
Translation in prokaryotes
Prokaryotic ribosome
(functional sites)
Peptidyl
Transferase
(rRNA 23S)
3’ end 16S rRNA
A-site: aminoacyl site
P-site: peptidyl site
E-site: exit site
fMet-tRNAiMet enters at the P site
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
Shine-Dalgarno consensus sequence
vs
Ribosome binding site
*
Procaryotic ribosomes initiate transcription at
ribosome-binding sites
Structure of a typical bacterial mRNA molecule
STOP
codon
STOP
codon
STOP
codon
Shine-Dalgarno sequences can be located anywhere (but specifically) along a mRNA molecule.
This permits bacteria to synthesize more than one type of protein from a single mRNA molecule
In prokaryotic cells,
transcription and translation take place simultaneously
An mRNA molecule may be transcribed simultaneously by
several ribosomes
Ribossomes
organized in
polysomes or
polyribosomes
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
Four steps involved in translation
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
fMet-tRNAiMet forms a complex
with IF-2 and GTP.
IF-2 directs initiator fMet-tRNAiMet
EF-1, blocks A site and is responsible for
conformational modification of small subunit
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
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
EF-G, mediates
translocation
The peptide bond
formation releases
the aa in the P site
from 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
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
Translation in eukaryotes
Key sites of interaction in the ribosome
Translation in eukaryotes
•
Efficient translation initiation also
requires the poly
poly--A 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 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
Two mechanisms of translation initiation
eIF-4G modified
version
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
Internal ribosome entry sites
The IRES-dependent mechanism requires only a
subset of the normal translation initiating factors,
and these assemble directly on the folded IRES
The initiation phase of protein synthesis in eucaryotes
eIF4E
eIF2 binds to tRNAiMet
eIF4A and eIF4B
have helicase activity
eEF-1, elongation
factor, similar to EF-Tu
eEF-2, translocation
factor, similar to EF-G
eRF-1 similar to tRNA and
recognizes termination codon
eRF-3 similar to bacteria RF-3
Regulation of gene expression at
translational level
– Translation initiation efficiency (includes RBS affinity
in prokaryotes)
– Coupling between transcription and translation (in
prokaryotes)
– Codon usage (codon preference or codon bias)
– mRNA degradation
Translational repression by antisense RNA in E. coli
OmpR (regulator)
micF
OmpF
OmpC
P
P
The micF RNA (mRNA interfering complementary RNA) is a translational repressor,
strongly complementary to the RBS-AUG region of the ompF mRNA.
The hybrid prevents ribosome binding and translation of ompF RNA
Ex. negative translational control in eukaryotes
•
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
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
Ferritin
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 receptor in order to
import less iron across the
plasma membrane