N-Formyl methionine

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... 

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. 

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 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... 

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 ... 

Protein synthesis in higher organisms is generally initiated by methionine. Bacteria, however, require N-formyl methionine. But studies on cells from higher forms of life indicate that the methionine is subsequently eliminated after incorporation of 15-20 amino acids. 

The biological importance of the second thiol amino add, homocysteine, is as the thioether and sulphonium ion derivatives. The free thiol occurs only as a metabolic intermediate. Methionine, the methyl thioether, is one of the twenty amino adds utilized for protein synthesis. Our concepts of the special significance of methionine in protein structure and function are only beginning to be developed, and will not be considered here. N-Formyl methionine also has the distinctive role of being a chain initiator in protein synthesis. The most extensively studied form of this thiol is S-adenosyl methionine or SAM, the sulphonium ion cofactor. This is the principal methylating reagent of biological systems and other alkyl transfers from the sulphonium ion are also known. 

The discrepancy between the in vivo and in vitro results with respect to the biosynthesis of F2 phage protein led to the discovery of the role of N-formyl methionine RNA in initiation of the polypeptide chain. When synthetic N-formyl methionine RNA is used in a cell-free system, it is found in the N-terminal position of the coat protein, and fingerprinting of the protein reveals that the N-terminal fragment includes an N-formyl methionine-alanine sequence. These findings suggest that in vivo methionine is split by a specific enzyme. Thus, the combination of in vivo and in vitro results suggests that the following sequence of events takes place in initiation. Some methionine molecules react with a special type of tRNA. The complex methionyl tRNA is accessible to an enzyme for formylation of the methionine. This methionyl tRNA has a special anticodon. The cell contains an enzyme that, at least in some cases, would split the formyl methionine from the polypeptide chain. 

Enzyme synthesis in vitro depends on the presence of N-formyl-methionine tRNA et. However, in systems prepared from normal cells there is no requirement for a formyl donor, since the cells contain an excess of the formyl donor N -formyltetrahydrofolic acid, stuck primarily to the ribosomes after disruption. Similar to the case with other tRNA species, the requirement for formylmethionyl can only be detected after special treatment of... 

Essential modification reactions of aminoacyl-tRNAs. In bacteria the initiator tRNA needed to start the synthesis of a polypeptide is initially aminoacyl-ated by methionine, but the metluonyl-tRN ArV1rl must then be N-formylated by transfer of a formyl group from N10-formyltetrahydrofolate (Fig. 15-18 ... 

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... 

Other kinds of modifications may be necessary to convert a newly synthesized protein to its biologically active form. The N-formyl group of the initiating methionine in prokaryotes is removed by a deformylase. A methionine amino-peptidase removes the initiating residue in many eukaryotic proteins. Other posttranslational modifications may include acetylation, amidation, hydroxy lation, methylation, phosphorylation, and sulfation of specific amino acid resi-... 

Most polypeptides synthesized on ribosomes are later chemically modified. Thus the formyl group on the N-terminal methionine in polypeptides of bacteria is removed by a deformylase. In both bacteria and eukaryotes, the N-terminal methionine, sometimes along with a few additional amino acids, is removed by aminopeptidases. 

The mechanism of N-acetylation of a-crystallin is quite interesting. The N-terminal residue has been identified as N-acetyl methionine. This methionine residue is derived from Met-tRNA et which is responsible for the initiation of the polypeptide chain and not Met-tRNAmet which incorporates the methionine residue in the growing polypeptide chain. It is clear that the N-acetylation is a true posttranslational process since acetyl Met-tRNA cannot replace formyl Met-tRNA et. Moreover, N-acetylation occurs when the polypeptide chain has reached a length consisting of approximately 25 amino acid residues. Other proteins, such as ovalbumin, are also acetylated during the early stages of polymerization on the polysome, and the protein acetyltransferase activity must therefore be associated with the protein-synthesizing apparatus. 

Formylation of methionylated tRNA " allows differentiation of the AUG start codon from internal AUG codons (14). MetRS aminoacylates tRNA" with methionine. A formyl group is linked covalently to the charged methionine via its amino moiety by the methionyl-tRNA formyltransferase enzyme, which uses N -formyl tetrahydrofolate as the formyl donor. This fMet-tRNA molecule binds directly to the P site of the ribosome to initiate protein synthesis, as compared with the A-site to which elongator tRNAs bind. 

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. 

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. 

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... 

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