Formyl methionine

The chemotactic peptides are also active in the stimulation of release of O and in the release of lysosomal contents Directed locomotion by PMNs is stimulated by a family of tri-, tetra-, and dipeptides which have in common formylated methionine as the first amino acid. Since formylated methionine is the initiator to which other amino acids are added in the synthesis of bacterial protein, small peptides which commence with formylated methionine are likely to be liberated proteolytically at sites of bacterial infection, possibly explaining their great potency as chematoxins. Using superoxide-dependent chemiluminescence (see below) as a measure of synthesis of O , Hatch et al. showed a hierarchy of potency of FMLP > FMP > FMV > FMA. Becker et al. measured the formation of O in suspensions of rabbit PMNs and found that the hierarchy of potency was the same for stimulation of the formation of O7 as it was for stimulation of release of lysosomal enzymes, namely... 

The distinction between an initiating (5 )AUG and an internal one is straightforward. In bacteria, the two types of tRNA specific for methionine are designated tRNAMet and tRNAfMet. The amino acid incorporated in response to the (5 )AUG initiation codon is A7-formyl-methionine (fMet). It arrives at the ribosome as A7-formylmethionyl-tRNAfMet (fMet-tRNAfMet), which is formed in two successive reactions. First, methionine is attached to tRNAfMet by the Met-tRNA synthetase (which in E. coli aminoacylates both tRNAfMet and tRNAMet) ... 

In all tRNAs the bases can be paired to form "clover-leaf" structures with three hairpin loops and sometimes a fourth as is indicated in Fig. 5-30.329 331 This structure can be folded into the L-shape shown in Fig. 5-31. The structure of a phenylalanine-carrying tRNA of yeast, the first tRNA whose structure was determined to atomic resolution by X-ray diffraction, is shown.170/332 334 An aspartic acid-specific tRNA from yeast,335 and an E. coli chain-initiating tRNA, which places N-formyl-methionine into the N-terminal position of proteins,336,337 have similar structures. These molecules are irregular bodies as complex in conformation as globular proteins. Numerous NMR studies show that the basic... 

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... 

Formyl methionine Proteolytic cleavage Protein aggregates Deamination... 

The problem of N-terminal variants in recombinant proteins is not uncommon. E. coli synthesizes proteins with a formylated methionine at the N terminus. In vivo, E. coli often removes N-formyl methionine with the action of a deformylase followed by methionine amino peptidase. This removal is not always exact and neighboring amino acids in the peptide chain influence the removal.130 This can yield recombinant products lacking a number of encoded amino acids at the N terminus. For smaller proteins, these product-related impurities generally are detected and quantitated by RP-HPLC. However, large proteins differing by only one or two N-terminal amino acids may be difficult to resolve by RP-HPLC. In these instances, peptide mapping by RP-HPLC is a valuable tool. 

Although the mechanism whereby proteins are synthesized is the same in all living forms, classical distinctions exist between eucaryal (post-transcriptional) and bacterial (co-transcriptional) translation. In-frame read-out of (usually polycistronic) bacterial mRNAs is established via Shine-Dalgarno mRNA 16S-rRNA recognition mechanisms polypeptide synthesis is initiated by a formylated methionine and the initiation reactions are assisted by a limited number of protein factors (three in Escherichia coli) that primarily influence kinetic parameters [3],... 

By contrast, eucaryal mRNAs are translated after extensive modifications of the primary transcripts that yield mature (generally capped and polyadenylated) monocistronic mRNAs. Recognition of translation start sites does not rely upon Shine-Dalgarno recognition instead, the small ribosomal subunit (generally) binds to the capped 5 end of mRNA and scans its nucleotide sequence until the initiator AUG codon is encountered. The polypeptide chains are initiated by a non-formylated methionine and the initiation reactions are aided by as many as 8-10 protein factors, some of which possess ATPase activity and perform functions not encountered in bacteria, such as cap recognition and mRNA unwinding (for a detailed review see ref [4]). 

One final interesting amino acid modification is that found in the methanogenic Archaea. These bacteria interpret the amber stop codon as the amino acid methylpyrrolysine, making this the 22" genetically encoded amino acid (the 21 being the N-formyl methionine that eukaryotes use to start translating all their proteins). 

The methionine residue found at the amino-terminal end of E. coli proteins is usually modified. In fact, protein synthesis in bacteria starts with N-jormylmethionine (fMet). A special tRNA brings formyl methionine to the ribosome to initiate protein synthesis. This initiator tRNA (abbreviated as tkNAf) differs from the tRNA that inserts methionine in internal positions (abbreviated as tRNAi O. The subscript f" indicates that methionine attached to the initiator tRNA can be formylated, whereas it cannot be formy-lated when attached to tRNA. Transfer RNAf can bind to all three possible initiation codons, but with decreasing affinity (AUG > GUG > UUG). In approximately one-half of E. coli proteins, N-formylmethionine is removed when the nascent chain exits the ribosome. 

A list of key differences between prokaryotes and eukaryotes with respect to protein synthesis is shown in Table 9-1. These include the existence of multiple eukaryotic initiation factors that facilitate the assembly of the riboso-mal protein synthetic machinery, whereas there are only three for prokaryotes. An initiation site on bacterial mRNA consists of the AUG initiation codon preceded with a gap of approximately 10 bases by the Shine-Dalgamo polypurine hexamer, whereas the 5 Cap (a 7-methylguanylate residue in a 5 —>5 triphosphate linkage) acts as an initiation signal in eukaryotes. In prokaryotes, the first or A-terminal amino acid is a formyl-methionine (fMet), but in eukaryotes it is usually a simple methionine. Additionally, the size and nature of the prokaryotic ribosomes are quite different from the eukaryotic ribosomes. 

The protein chain grows in a particular direction, so that the first amino acid of the sequence has a free NH2 group whilst the last amino acid has a free COOH. Experiments have shown that in most cases the N-terminal amino acid is methionine. So Met-t-RNA would seem to be the initiator. But methionine can also be inserted at other positions in the chain, so how does Met-t-RNA know where to place its amino acid It is not codon directed, as there is, it is apparent from Figure 23, only one trinucleotide sequence corresponding to methionine, the AUG triplet. In bacteria at least (in which most of these experiments have been carried out), it seems that to serve as an initiator, the methionine must be converted to the unusual amino acid formyl-methionine for attachment to the P site. It seems probable that a similar mechanism exists in mammalian cells although the position here is not yet completely clear. 

So once the formyl-methionine initiator is in place, attached to the m-RNA on the A site of the ribosome, chain elongation... 

The 1-Fe rubredoxins are single polypeptide chain proteins of about 55 amino acid residues and 6,000 dalton molecular weight. The primary structure of the Microccus aerogenes (220, 221) and Peptostreptoccus elsdenii (210) proteins have been determined. The two amino acid sequences (Fig. 11) reveal a relatively high mutation frequency, with a noticeable conservancy around the four cysteinyl residues which are critically involved in binding the iron and hence in the proposed electron transfer role of the protein (210). Another curious feature of the anaerobic rubredoxins is the presence of N-formyl methionine as N-terminus amino acid (222). Synthesis of the polypeptide is in progress (223). 

Two types of acetylated amino acid residues occur in proteins. The N-terminal amino acid residue in a protein may be acetylated following proteolytic removal of the terminal methionine (eukaryotes) or a-N-formyl methionine (prokaryotes) residue subsequent to translation. Data... 

A 12-nucleotide deletion (positions 1219-1230) observed within an rRNA hairpin structure between nucleotides 1198 and 1247 in domain II of the E. coli 23S rRNA gene conferred erythromycin resistance. This 12-nucleotide sequence is located upstream of an open reading frame (ORE), which encodes a peptide MVLFV, E-peptide [190]. The conserved amino acids required for forming specific contacts with rRNA or ribosomal proteins may be N-terminal formyl methionine, the third Leu or lie, and C-terminal hydrophobic amino acid commonly represented by Val [190]. The expression of the pentapeptide in vivo renders E. coli cells resistant to erythromycin [167]. Curiously, such a deletion and... 

In the genetic code, methionine is coded for by the codon AUG. This codon is called the start codon because methionine is the first amino acid used to build a protein chain. Methionine forms the so-called amino terminus of a protein. In prokaryotes, a modified form of methionine, formyl-methionine is used as the first (but not subsequent) amino acid in proteins. Formyl-methionine is carried by a modified tRNA from the tRNA that carries unmodified methionine. 

Lohse and Szostak [13] used a selection scheme that mimicks the transfer of formyl-methionine from a fragment of fMet-tRNA onto the hydroxy group of hydroxypuro-mycin, a simplified version of the ribosomal peptidyltransferase reaction. An RNA library... 

The information for the amino acid sequence is contained in the mRNA. Synthesis commences at the correct codon of the mRNA because a special amino acid derivative, N-formyl methionine ... 

First amino acid Methionine Formyl-methionine... 

Fig. 15.9. Bacterial tRNA containing formyl-methionine. The initial methionine is not formylated in eukaryotic protein synthesis.

---