Nascent polypeptides bear the N-terminal Met residue, encoded by the AUG initiation codon. In bacteria and in eukaryotic organelles mitochondria and chloroplasts (remote descendants of bacteria), this Met is Nα-terminally formylated (N-formylated) through a “pretranslational” mechanism. Formyltransferase (FMT) uses 10‑formyltetrahydrofolate to formylate the α-amino group of the Met moiety in the initiator tRNAiMet.
This transformylase is more selective than the Met-tRNA synthetase, and it cannot formylate free methionine or Met residues attached to tRNAMet. Instead, it is specific for Met residues attached to tRNAfMet, presumably recognizing some unique structural feature of that tRNA. The other Met-tRNA species, Met-tRNAMet, is used to insert methionine in interior positions in the polypeptide chain. Blocking of the amino group of methionine by the N-formyl group not only prevents it from entering interior positions but also allows fMet-tRNAfMet to be bound at a specific initiation site on the ribosome that does not accept Met-tRNAMet or any other aminoacyl-tRNA.
The resulting formyl-Met (fMet) becomes the first residue of a nascent polypeptide that emerges from a bacterial ribosome.
The formyl moiety of N-terminal fMet is cotranslationally removed by peptide deformylase (PDF), which is reversibly bound to the ribosome near the exit from the ribosomal tunnel.
Once N‑terminal fMet of a nascent protein is deformylated by PDF, the resulting Met can be cleaved off by Met‑aminopeptidase (MetAP). The removal of (deformylated) Met by MetAP requires that a residue at position 2, to be made N‑terminal by the cleavage, is not larger than Val.
This sequence of addition and removal appears futile, yet every sequenced bacterial genome encodes the enzymes for formylation and deformylation, suggesting this process is essential for some reason. Mitochondrial translation in yeast does not require FMT1, the yeast methionyl-tRNA formyltransferase homologue. It has been like a puzzling paradox, it is found but is not necessary for the process, yet it has not been removed during evolution.
Despite being ubquitous, formylation is not an essential activity in most bacteria. Formylation can be inhibited through the action of the antibiotic trimethoprim. Trimethoprim prevents tetrahydrofolate (THF) production through inhibition of dihydrofolate reductase, consequently preventing production of the formylase cofactor 10-CHO-THF.
Consequently, translation proceeds with non-formylated Met-tRNAifMet – similar to translation in eukaryotes and archaea, albeit with a reduced growth rate. In the absence of formylase activity, the deformylase gene can also be deleted.
“What, then, is the main biological function of this metabolically costly, transient, and not strictly essential modification of N‑terminal Met, and why has Met formylation not been eliminated during bacterial evolution?”
Following are the hypotheses:
- It has been hypothesised that formylation of Met-tRNAi fMet allows discrimination between initiator and elongator tRNAs at the ribosome. Indeed, IF2 has a higher affinity for fMet-tRNAi fMet than Met-tRNAi fMet due to electrostatic interactions in the IF2 initiator tRNA binding cleft.
- A second hypothesis for the function of formylation is that it prevents unfavourable side reactions involving the reactive amino group of methionine. Indeed, formylation of Met-tRNAi fMet does reduce the sensitivity of the aminoacyl-linkage to hydrolysis.
- A third hypothesis for the function of formylation considers the expression of polycistronic mRNA in bacteria. It is proposed that the difference in utilisation of formylation between bacteria and eukaryotes mirrors the use of polycistronic mRNA in these domains. That is, bacteria utilise formylation and transcribe many genes in polycistronic messages, whereas eukaryotes do not use formylation and almost always use monocistronic mRNAs
- All of the currently proposed theories on the function of Met-tRNAi formylation have been developed from an ‘adaptionist’ viewpoint. That is, the presence of formylation has been explained by attempting to attribute a fitness advantage to cells utilizing the process.
- The formylated N-terminal fMet can act as a degradation signal, largely a cotranslational one. One likely function of fMet/N-degrons is the control of protein quality. In bacteria, the rate of polypeptide chain elongation is nearly an order of magnitude higher than in eukaryotes. The faster emergence of nascent proteins from bacterial ribosomes is one mechanistic and evolutionary reason for the pretranslational design of bacterial fMet/N‑degrons, in contrast to the cotranslational design of analogous Ac/N‑degrons in eukaryotes.
The fact that formylation is not necessary for translation in any of the three domains of life (it is absent from eukaryotes and archaea and dispensible from most bacteria) suggests it is not a critical feature of translation initiation. A model of the evolution (Catchpole, Ryan Joseph) of formylation resembles theories of constructive neutral evolution. Constructive neutral evolution describes how complexity can arise without providing an adaptive advantage to hosts. There is still a lot, yet to be found and discovered.