Translation in Prokaryotes

(Cooper, 1997 p. )

The process that uses the base sequence in mRNA to synthesize a polypeptide with "complementary" amino acid sequence.

As with the synthesis of RNA we also have a directed synthesis for polypeptides: The information in mRNA is always read from the 5' to the 3' direction. The polypeptide is synthesized from its amino terminus to its carboxyl terminus.

RNA: 5'----------------------------------3' polypeptide: H2N-----------------------------COOH

The translation process occurs at the ribosome and involves the three major classes of RNA: mRNA, tRNA, and rRNA. This is true for prokaryotes and eukaryotes.

Earlier in the semester we talked about the genetic code, about tRNAs and how they are specifically loaded with the correct amino acids and about ribosomes and their two specific subunits.

One more important finding:
Organisms usually do not contain as many different kinds of tRNAs as there are codons for amino acids (remember: 64 possible combinations for a triplet formed from 4 different bases (4 to the 3). 3 of these combinations are stop codons, but 61 code for amino acids).
In E. coli for example there are about 40 different tRNAs for the 20 different amino acids. It turns out that some tRNAs can base pair with more than one codon so all 61 amino acid codons are recognized.
The codon-anticodon base pairing requirements are somewhat less stringent than those for DNA or RNA synthesis. This was first proposed by Francis Crick in 1965 and became known as the wobble hypothesis. Crick proposed that the conformational constraints for codon-anticodon base pairing are less stringent because they do not occur in the context of a double helix which is so easily distorted.

For example G-U base pairing can occur in codon-anticodon interaction (Fig. 7.3).

Wobbling (the less stringent, unusual base pairing due to conformational relaxation) usually occurs at the third base in a triplet codon (which corresponds to the first base in an anticodon).

So how does translation occur? Via the 3 phases initiation, elongation and termination (Fig. 7.8).

PROKARYOTIC TRANSLATION

In E. coli and other prokaryotes the mRNA is transcribed without prior processing. But translation does not start directly at the 5' end of the mRNA. Instead we find an untranslated region at the 5' end called 5' untranslated region or 5' UTR.

Translation always starts with the amino acid methionine and therefore usually at an AUG codon: All newly synthesized proteins contain a methionine at their amino terminus (often cleaved off after synthesis).
In E. coli and many other prokaryotes the tRNA for methionine at start of translation different from the tRNAMet used in the middle of the coding sequence: specific initiator tRNA. And additionally the methionine contained in the initiator tRNA is modified, it contains a formyl group at its amino (N) group (N-formylmethionine): tRNAfMet.

In prokaryotic mRNA, the first AUG at the 5' end is not necessarily the start site for translation. Instead the start codon is always preceeded by a sequence specifying the AUG as start site. The specific sequence (AGGAGGU) is called Shine-Dalgarno sequence after its discoverers. The sequence base pairs with a complementary sequence near the 3' terminus in the 16S rRNA (part of the small, 30S ribosomal subunit). This specific base pairing alines the mRNA and the ribosome.

In initiation (Fig. 7.9):

First the 70s ribosome must separate into 50s and 30s subunits.
The 3 initiation factors IF-1, IF-2, and IF-3 then bind to the 30S ribosomal subunit.
Then the specific initiator tRNA carrying the N-formylmethionine and the mRNA join the complex (remember base pairing via Shine-Dalgarno sequence). The initiator tRNA is specifically recognized by IF-2.
At this point in the process we have what is termed a 30s preinitiation complex.
The 50S subunit binds, triggering the release of the IFs.

Thus, formation of the 70S initiation complex is completed and it is ready for the next phase, elongation.

In elongation (Fig. 7.11):

The first step is the binding of a second charged tRNA that base pairs specifically with the second codon. EF-Tu and GTP bind the charged tRNA and help to align the tRNA in the A site. When base pairing of the anticodon-codon occurs, GTP is hydrolyzed and EF-Tu + GDP are released.

The binding of the tRNA to the ribosome is possible because

the large subunit of the ribosome contains several sites that tRNAs can occur in and bind to by its D loop and T psi C loop:

the P(peptidyl) site occupied at the moment by the initiator tRNA,
the A (aminoacylor acceptor) site occupied at the moment by the second tRNA,
and an E (exit) site currently unoccupied.

The second step is the formation of a peptide bond between the two amino acids, a process catalyzed by the enzyme peptidyl transferase which is associated with the large ribosomal subunit. The result is a transfer of the N-formylmethionine to the second tRNA in the A site which carries now a dipeptide and the initiator tRNA being uncharged in the P site.

The third step is the translocation of the ribosome to move 3 bases or 1 codon along the mRNA. This translocation also positions the initiator tRNA to the E or exit site, the second tRNA with the dipeptide to the P site and leaves the A site unoccupied but with the third codon exposed. Translocation is catalyzed by the elongation factor EF-G. The G indicates that this factor uses the energy gained from the hydrolysis of GTP for translocation to occur.

Finally a third tRNA is escorted by EF-Tu to the A site and the anticodon base pairs specifically with the codon. This binding triggers the release of the initiator tRNA from the E site and allows the complex to be ready for another round of peptide bond formation, translocation and codon-anticodon base pairing (and so on).

Termination of translation (Fig. 7.13)

Begins when a stop codon appears after translocation in the A site of the ribosome (UAA, UAG, UGA).

There is no tRNA that can base pair with the stop codons. Instead release factor RF-1 specifically binds to UAA or UAG in the A site and RF-2 specifically binds to UGA.

Both release factors if inserted cause the hydrolysis of the bond between the tRNA in the P site and the polypeptide chain that is attached to it: The result is the release of the newly synthesized polypeptide.
This release in turn causes the complex to dissociate and mRNA, tRNA and ribosomal subunits are freed.

Because the stop codon never occurs at the very end of the mRNA, we find a 3' untranslated region or 3'UTR.

A single mRNA molecule is not only translated once. In fact as soon as the ribosome has moved away from the initiation site, another round of initiation can begin (Fig. 7.14). It means that a single mRNA is often transcribed by many ribosomes at the same time, usually 100 to 200 bases apart from each other. Such a group of ribosomes on the same mRNA is called a polyribosome or polysome. However, at each single ribosome a polypeptide is synthesized independently.

To fully understand the way translation occurs in E. coli, we have to remember two additional things:

prokaryotic mRNAs are often polycistronic
in prokaryotes there is no nucleus that separates transcription from translation spatially

Polycistronic mRNA means that it contains the coding region for several polypeptides (Fig. 7.6). In order for each coding region to be translated, it has to have all the specificities for translation: a Shine-Dalgarno sequence in front of an AUG start codon and a stop codon.

No nucleus for spatial separation between transcription and translation in prokaryotes and
translation starts near the 5' end of the mRNA (the end that is first synthesized in transcription)

--------- translation can be initiated even before the mRNA is fully synthesized .

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

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