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

Protein Organism

Prokaryotic enzymes

Formate dehydrogenase

Clostridium thermoaceticum, Clostridium thermoautotrophicum, Enterobacter aerogenes, Escherichia coli, Methanococcus vaniellii

Proteins containing selenocystein

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

3’

5’

3’

5’

fMet-tRNAiMetMet-tRNAMet

A*

UAC

AUG

A*- adenosina alquilada

3’5’

A

AAC

GUG3’5’

A não modificação da adenosina

a 3’ do anticodão do tRNAi permite

uma certa flexibilidade de

emparelhamento do fMet-tRNAiMet

3’

5’

3’

5’

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)

Genomes 11.5

to N-formylmethionine (transformilase)

Bacteria IF2 only recognizes fMet-tRNAiMet

Eucaryotic eIF2 only recognizes Met-tRNAiMet

Translation in prokaryotes

Prokaryotic ribosome(functional sites)

Peptidyl

Transferase

(rRNA 23S)

3’ end 16S rRNA

fMet-tRNAiMet enters at the P siteA-site: aminoacyl site

P-site: peptidyl site

E-site: exit 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

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

Ribossomes

organized in

polysomes or

polyribosomes

Four steps involved in translation

Dynamic

equilibrium

INITIATION of translation in bacterial cells requires several

initiation factors and GTP

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 GDP and the large subunit joins to

create the 70S initiation complex

The ELONGATION of translation comprises three steps

Complex EF-Tu, EF-Ts,

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 UAA UAG

RF-2 UAA UGA

RF-3 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 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 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

Internal ribosome entry sites

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

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

eIF2 binds to tRNAiMet

eIF4E

eIF2 binds to tRNAiMet

eIF4A and eIF4B

have helicase activity

eEF-2, translocationfactor, similar to EF-G

eEF-1, elongationfactor, similar to EF-Tu

eRF-1 similar to tRNA and

recognizes termination codon

eRF-3 similar to bacteria RF-3

Regulation of gene expression at

translational leveltranslational level

– Translation initiation efficiency (includes RBS affinity

in prokaryotes)

– Coupling between transcription and translation (in

prokaryotes)prokaryotes)

– Codon usage (codon preference or codon bias)

– mRNA degradation

Translational repression by antisense RNA in E. coli

micF OmpC

P

P

OmpR (regulator) OmpF

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

Transferrin receptor and ferritin are regulated

by different types of mechanisms, their levels

IRE- iron response element

Ferritin Transferrin receptor

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

by different types of mechanisms, their levels

respond oppositely to iron concentrations

even though they are regulated by the same

iron-responsive regulatory protein