N-Formylmethionine

It has long been known that peptides of bacterial origin, such as N-formylat-ed oligopeptides, are potent activators of neutrophils. Bacterial protein biosynthesis is initiated by the codon AUG, which codes for polypeptide chains at the NH2 terminus to start with N-formylmethionine. However, very few mature bacterial proteins actually have this amino acid at the NH2 terminus because Af-formylmethionine is cleaved off by proteolytic processing. Sometimes just this amino acid is cleaved, but often several adjacent residues are also removed with it. These observations formed the basis for the chemical synthesis of a variety of N-formylated oligopeptides and an assessment of their ability to activate neutrophils in vitro. The most potent of these formylated peptides is TV-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). 

The first phase of translation, initiation, involves several steps. First, two proteins, initiation factors IF-1 and IF-3, bind to the 30 S subunit (1). Another factor, IF-2, binds as a complex with GTP (2). This allows the subunit to associate with the mRNA and makes it possible for a special tRNA to bind to the start codon (3). In prokaryotes, this starter tRNA carries the substituted amino acid N-formylmethionine (fMet). In eukaryotes, it carries an unsubstituted methionine. Finally, the 50 S subunit binds to the above complex (4). During steps 3 and 4, the initiation factors are released again, and the GTP bound to IF-2 is hydrolyzed to GDP and Pj. 

In E. coli polypeptide chains are always initiated with the amino acid N-formylmethionine. Some bacteria can apparently live without the ability to formylate methionyl-tRNA,290 but most eubacteria as well as mitochondria and chloroplasts use formyl-methionine for initiation. In a few cases, both among bacteria and eukaryotes, initiation can sometimes occur with other amino acids 291 The first step is the alignment of the proper initiation codon correctly on... 

Eukaryotic ribosomes are larger (80S) and more complex than prokaryotic ribosomes (70S). Initiation is basically similar in prokaryotes and eukaryotes except that in eukaryotes at least nine initiation factors are involved (cf. three factors in prokaryotes), the initiating amino acid is methionine (cf. N-formylmethionine in prokaryotes), eukaryotic mRNAs do not contain Shine-Dalgarno sequences (so the AUG initiation codon is detected by the ribosome scanning instead), and eukaryotic mRNA is monocistronic (cf. some polycistronic mRNAs in prokaryotes). Initiation in eukaryotes involves the formation of a 48S preinitiation complex between the 40S ribosomal subunit, mRNA, initiation factors and Met-tRNA 61. The ribosome then scans the mRNA to locate the AUG initiation codon. The 60S ribosomal subunit now binds to form the 80S initation complex. 

A special initiator tRNA, tRNAme i (I stands for initiator) is used for beginning protein synthesis. In bacteria, this initiator tRNA carries the modified amino acid N-formylmethionine (fmet). The formylation reaction transfers the formyl group from formyl-tetrahydrofolate to... 

Proteins are typically made as pro-proteins and are then subsequently modified by post-translational processing involving selective proteolysis ( trimming ) and addition of other groups. Thus, nascent polypeptides commence with N-formylmethionine (bacteria) or methionine (eukaryotes). However, N-terminal sequences are often removed in proteolytic processing. In many eukaryote proteins, the final N-terminal amino acid of the processed protein is N-acetylated. The C-terminus may also be changed by peptide cleavage and other covalent modification. 

The methionine residue found at the amino-terminal end of E. coli proteins is usually modified. In fact, protein synthesis in bacteria starts with H-formylmethionine (fMet). A special tRNA brings formylmethionine to the ribosome to initiate protein synthesis. This initiator tRNA (abbreviated as tRNAf) differs from the one that inserts methionine in internal positions (abbreviated as tRNA ). The subscript "f indicates that methionine attached to the initiator tRNA can be formylated, whereas it cannot be formyl-ated when attached to tRNA. In approximately one-half of E. coli proteins, N-formylmethionine is removed when the nascent chain is 10 amino acids long. 

Using partial structural formulas, describe the reactions by which serine and methionine react to form N-formylmethionine needed for protein synthesis. 

Fig. 12.4 Outline of the process of protein synthesis (translation of messenger RNA) in bacterial cells. The four stages of synthesis are shown initiation, elongation, translocation and termination with the sites of action of antibiotics. AUG is the start codon on messenger RNA (mRNA) specifying the first amino acid in bacterial proteins, N-formylmethionine. UAG, UAA and UGA are termination codons specifying no amino acid. 30S and 50S are the subunits of the ribosome. Other protein factors involved in protein synthesis are initiation factors (IF-1,2,3), elongation factors (EF-Tu and EF-G) and release factors (RF-1,2,3).

Cech s group was the first to have success in this direction (Piccirilli, 1992). Using a genetically modified Tetrahymena ribozyme, they were able to hydrolyse an ester bond between the amino acid N-formylmethionine and the corresponding tRNAf-Met. The reaction was, however, very slow, only about 5 to 15 times faster than the uncatalysed reaction. The authors ventured to suggest that these ribozymes could have functioned as the first aminoacyl tRNA synthetases. 

A single tRNA specifies both methionine tRNA and N-formylmethionine tRNA by seeondary modification of the primary transcript. 

The codon AUG has two functions. It corresponds to the amino acid methionine when AUG occurs within a coding sequence in the mRNA, i.e., within a polypeptide chain. It also serves as a signal to initiate polypeptide synthesis—with methionine for eukaryotic cells but with N-formylmethionine for prokaryotic cells. How the protein-synthesizing system distinguishes an initiating AUG from an internal AUG is discussed below. The codon GUG also has both functions, but it is only rarely used in initiation. Once initiation has occurred at an AUG codon, the reading frame is established and the subsequent codons are translated in order. 

N-formylmethionine (fMet) Transformylase does not formylate methionyl tRNA because tRNA and tRNA are structurally different. A second feature is its ability to initiate polypeptide synthesis. 

The initiator tRNA carries an N-formylmethionine. The formyl group is added after the methionine is linked to the tRNA by an enzyme called transformylase. It transfers a formyl group from NiO-formyltetrahydrofolate. 

Only tRNAf is accepted to form the initiation complex. All further charged tRNAs require fully assembled (i.e., VOS) ribosomes. All prokaryotic proteins are synthesized with the same N-terminal residue, N-formylmethionine. 

For human beings methionine is nutritionally essential and comes entirely from the diet. ITowever, the oxoacid analog of methionine can be used as a nutritional supplement. Dietary homocysteine can also be converted into methionine to a limited extent. Methionine is incorporated into proteins as such and as N-formylmethionine at the N-terminal ends of bacferial profeins (steps a and b, Fig. 24-16). In addition to its function in proteins methionine plays a major role in biological methylation reactions in all organisms. It is converted into S-adenosylmethionine (AdoMet or SAM Fig. 24-16, step e see also Eq. 17-37), ° ° which is the most widely used methyl group donor for numerous biological methylation reactions (Eq. 12-3). S-Adenosylmethionine is also the precursor of the special "wobble base" queuine (Fig. 5-33). The product of transmethylation, S-adenosylhomocysteine, is converted (step... 

A tRNA with an attached N-formylmethionine forms hydrogen bonds with the codon. 

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