Formylmethionine

FIGURE 28.12 Translation of rnRNA to an amino acid sequence of a protein starts at an rnRNA codon for methionine. Nucleophilic acyl substitution transfers the A/-formylmethionine residue from its tRNA to the amino group of the next amino acid (shown here as alanine). The process converts an ester to an amide. 

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 A-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. 

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). 

McLeish, K. R., Gierschik, P., Schepers, T., Sidiropoulos, D., Jakobs, K. H. (1989). Evidence that activation of a common G-protein by receptors for leukotriene B4 and IV-formylmethionine-leucyl-phenylalanine in HL-60 cells occurs by different mechanisms. Biochem. J. 260, 427-434. 

There is one start codon (initiation codon), AUG, coding for methionine. Protein synthesis begins with methionine (Met) in eukaryotes, and formylmethionine (finet) in prokaryotes. 

The mRNA is bound to the smaller 30S subunit of the bacterial ribosome. The mRNA is a transcription of one of the genes of DNA, and carries the information as a series of three-base codons. The message is read (translated) in the 5 to 3 direction along the mRNA molecule. The aminoacyl-tRNA anticodon (UAC) allows binding via hydrogen bonding to the appropriate codon (AUG) on mRNA. In prokaryotes, the first amino acid encoded in the sequence is A-formylmethionine (fMet). Although the codon for initiation (A-formylmethionine) is the same as... 

Some special features of proteins are elaborated by secondary transformations that are not part of the translation process. The A-formylmethionine initiator may be hydrolysed to methionine, or, as we have already indicated, the methionine unit may be removed altogether. Other post-translational changes to individual amino acids may be seen, e.g. the hydroxylation of proline to hydroxyproline (see Section 13.1) or the generation of disulfide bridges between cysteine residues (see Section 13.3). 

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. 

How can the single (5 )AUG codon distinguish between the starting At-formylmethionine (or methionine, in eukaryotes) and interior Met residues The details of the initiation process provide the answer. 

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... 

Formation of the first peptide linkage. The formylmethionine group is transferred from its tRNA at the P site to the amino group of the second aminoacyl-tRNA at the A site of the ribosome. This involves nucleophilic attack by the amino group of the second amino acid on the carboxyl carbon of the methionine. The resulting bond formation attaches both amino acids to the tRNA at the A site. 

Despite the fact that only 20 amino acids (plus selenocys-teine and formylmethionine in prokaryotic systems) are known to be directly specified by the genetic code, chemical analysis of mature proteins has revealed hundreds of different amino acids, all of them structural variants on the original 20. This structural diversity, which greatly expands the chemical lexicon of proteins, results from posttranslational modification of the primary products of translation. Our knowledge of the nature and significance of enzymatic reactions that bring about these important alterations is still very incomplete. 

FIGURE 3.7 A cryo-EM map of the Escherichia coli ribosome (complexed with fMet-tRNAf Met and mRNA) where fMet = formylmethionine obtained from 73,000 particles at a resolution of 11.5 A. (a-d) Four views of the map, with the ribosome 30S subunit painted in yellow, the ribosome 50S subunit in blue, helix 44 of 16S RNA in red, and fMet-tRNA at the P site in green. Inset on top juxtaposes the experimental tRNA mass (green, on left) with the appearance of the X-ray structure of tRNA at 11 A resolution (on right). Arrows mark points at which tRNA contacts the surrounding ribosome mass. Landmarks h = head and sp = spur of the 30S subunit. CP = central protuberance LI = LI stalk and St = L7/L12 stalk base of the 50S subunit. 

A Yoshida, M Lin. NH2-terminal formylmethionine- and NH2-terminal methioninecleaving enzymes in rabbits. J Biol Chem 247 952-957, 1972. 

The first codon translated in all mRNAs is AUG which codes for methionine. This AUG is called the start codon or initiation codon. Naturally, other AUG codons also occur internally in an mRNA where they encode methionine residues internal to the protein. Two different tRNAs are used for these two types of AUG codon tRNAfMet is used for the initiation codon and is called the initiator tRNA whereas tRNAmMet is used for internal AUG codons. In prokaryotes the first amino acid of a new protein is /V-formylmethionine (abbreviated fMet). Hence the aminoacyl-tRNA used in initiation is fMet-tRNAfMet. It is essential that the correct AUG is used as the initiation codon since this sets the correct reading frame for translation (see Topic HI). A short sequence rich in purines (5 -AGGAGGU-3 ), called the Shine-Dalgarno sequence, lies 5 to the AUG initiation codon (Fig. 3) and is complementary to part of the 16S rRNA in the small ribo-somal subunit. Therefore this is the binding site for the 30S ribosomal subunit... 

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... 

Peptide deformylase (PDF), which is not found in mammalian cells, is an essential bacterial met-alloenzyme that has received much attention as one of the targets for developing novel antibiotics (Clements et al., 2001 Hackbarth et al., 2002). Bacterial protein synthesis, unlike cytosolic protein synthesis in mammalian cells, is initiated by A-formylmethionine (Adams and Capecchi, 1966). 

The /V-formylmethionine of a nascent protein synthesized in bacteria is removed by the sequential activities of PDF and a methionine aminopeptidase to generate the mature protein. The gene encoding PDF was cloned and overexpressed in E. coli by Meinnel and coworkers (1993). The PDF enzyme has an unusual metal ion (Fe2+) as its catalyst. However, the ferrous ion in this enzyme is unstable and can be quickly and irreversibly oxidized to ferric ion, rapidly inactivating the enzyme. PDF-based assay development therefore depended on the ability of nickel ion to replace ferrous ion in vitro, increasing the stability of the enzyme and maintaining its enzymatic activity (Groche et al., 1998 Clements et al., 2001 Hackbarth et al., 2002). 

The first amino acid for reaction is jV-formylmethionine (fMet) which has a specific tRNA (tRNA 1 1) (as opposed to the Met-specific tRNA tRNAmMl 1). Using GTP hydrolysis as an energy source, the 30S subunit complexes with initiation factors IF1, IF2 and IF3. This complex binds the mRNA with the anticodon (3 -UAC-5 ) of the jV-formylmethionyl-tRNA M t (fMet-tRNAf 11 1) hydrogen bonding to the start codon (5 -AUG — 3 ) of the mRNA, the fMet-tRNA -Ml 1 binding at the so-called P site with release of IF3. 

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. 

F26BP, fructose-2,6-bisphosphate FA, fatty acid FAD, fatty acid desaturase FADH2/FAD, reduced/oxidized flavin adenine dinucleotide F -ATPase, ATP synthetase F complex FGF, fibroblast growth factor FGF-RTK, fibroblast growth factor receptor tyrosine kinase Fmet, formylmethionine FMNH2/FMN, reduced/oxidized flavin mononucleotide... 

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