Translation in Eukaryotes

(Cooper, 1997 p. )

EUKARYOTIC TRANSLATION Eukaryotic primary transcript is processed extensively.
Two major differences in prokaryotic mRNA and eukaryotic mRNA are the presence of the 5' CAP and the 3' poly(A)tail. The 5' CAP of 7-methyl guanosine is attached by an unusual 5'-5' linkage to the triphosphate of the first ribonucleotide.

See figure

As we will soon see, the 5' CAP plays an important role in translation.

In initiation: there are at least 10 different eukaryotic initiation factors (designated eIFs). Recall that there are only 3 known IFs in prokaryotic translation.

In elongation: eukaryotic elongation factor eEF-1alpha, eEF-1betagamma, eEF-2

In termination: single eukaryotic release factor eRF

second difference from prokaryotes: initiation with unmodified methionine (but also at AUG codon and also with specific initiator tRNA)

Greatest difference to prokaryotes: initiation (no Shine-Dalgarno sequence, no initial base pairing between mRNA and rRNA)

Initiation starts (Fig. 7.10)

eIFs bind not only to the small ribosomal subunit (now of course the 40S ribosomal subunit), but some other eIFs bind to mRNA and another eIF binds to the initiator tRNA. The 5' cap of mRNA is important because the specific eIFs that bind to mRNA recognize and associate with the 5' cap[Also termed CAP binding proteins]. The single eIF that associates with the initiator tRNA carries a GTP (important later).
The eIFs of mRNA and the initiator tRNA bring them to the 40S ribosomal subunit: the 40S subunit first associates with the very 5' end of mRNA.

Then, the ribosomal subunit, in association with eIFs and initiator tRNA, scans the mRNA until it reaches the AUG start codon. Scanning requires energy and is accompanied by ATP hydrolysis. It is not always the first AUG that is recognized as a start codon, sequences around first AUG might reduce efficiency of initiation so scanning continues to next AUG.

When AUG start codon reached, the GTP associated with the eIF of initiator tRNA is hydrolyzed. This causes all eIFs to be released from complex. Then the large (60S) ribosomal subunit can bind.

Therefore, a 80S initiation complex is formed.

Elongation: very similar to prokaryotes

eEF-1a same function as EF-Tu (escorting charged tRNA to A site in ribosome, carrying GTP that is hydrolyzed before release of eEF-1a, slow hydrolyzation serves proofreading)
eEF-2 same function as EF-G (promotes translocation of ribosome, accompanied by GTP hydrolysis)
eEF-1 beta gamma has the same function as EF-Ts (regenerates used eEF-1a via exchange of GDP for GTP)

Termination: very similar to prokaryotes
only difference: single release factor (eRF) recognizes all three stop codons.

Eukaryotic mRNA:

never polycistronic.
Initiation of translation can only occur after mRNA transported from nucleus to cytoplasm (ribosomes only in cytoplasm). Transport only of fully processed mRNA.

But: polysomes also in eukaryotes.

Proof-Reading During Translation

Several amino acids are structurally similar. It is to be expected that amino acyl synthetases might make occasional mistakes.

Example: valine and isoleucine constitute a pair of potentially ambiguous amino acids. In fact, isoleucyl synthetase makes this error at a frequency of 1/225 activation events.

There is, however, a built-in editing mechanism. The incorrect amino acid is removed from the tRNA and the hydrolysis is catalyzed by the amino acyl synthetase.

Regulation of translation:
Important in both prokaryotes and eukaryotes (although regulation via transcription even more important)

Example for one mechanism:
binding of repressor protein to specific sequence in particular mRNA
---------- translation inhibited

specific example for regulation via repressor protein (Fig. 7.15):
ferritin (protein that stores iron in cell)

much iron: much ferritin needed
little iron: little ferritin needed

specific repressor protein for ferritin mRNA:

much iron: repressor binds iron, ferritin mRNA is translated, much ferritin
little iron: repressor cannot bind iron, instead binds to specific RNA sequence in ferritin mRNA called iron response element (IRE), ferritin mRNA is not translated, little ferritin

References:
Cooper, Geoffrey M. (1997) The Cell: A Molecular Approach; ASM Press, Washington, D.C. / Sinauer Associates, Inc., Sunderland, MA.