Formyl methionyl tRNA

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... 

Translation starts with 7V-formyl-methionyl-tRNA [32] + - - - -... 

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. 

Formylated methionyl-tRNA is important for initiation of translation Single mRNAs specify more than one gene product Cycloheximide blocks elongation during translation Cytosolic ribosomes are smaller than those found in prokaryotes Erythromycin inhibits elongation during translation... 

Linezolid inhibits initiation by blocking formation of the N-formyl-methionyl-tRNA-ribosome-mRNA I... 

Linezolid is an oxazolidinone that inhibits the formation of the initiation complex in bacterial translation systems bv preventing formation of the JV-formyl-methionyl-tRNA-rihosome-mRNA ternary complex. 

Fig. 2. Formation of a stable initiation complex between a 70 S ribosome and messenger RNA. In the final complex fMet-tRNAf " is in the correct position for the formation of a peptide bond. IF-1, IF-2, and IF-3 are the protein initiation factors and fMet-tRNAf " is the formyl methionyl tRNA which is used for the initiation of protein synthesis in prokaryotes. The process in animal cells is thought to be substantially the same, the initiation factors being termed IF-Ml, IF-M2, and IF-M3 and the initiator tRNA, Met-tRNAt . The methionine attached to this tRNA species is not normally formylated but can be so modified by enzymes from bacterial cells.

Further studies of the N-terminal amino acid of ribosomal and soluble proteins of E. coli indicated that 90% of these proteins end with methionine. Alanine, serine, and threonine were the N-terminal amino acids in the other proteins. In spite of the great uniformity in N-terminal amino acids, the amino acid composition of these proteins was found to be quite heterogeneous. Studies of the codon of the methionyl tRNA that can be formylated and of the methionyl tRNA that cannot be formylated indicated that AUG functions as a codon for both. A small amount of methionyl tRNA that could be formylated was also coded by GUG and UUG. The fact that AUG codes for both methionyl tRNA s is not irreconcilable with an eventual role of N-formyl methionyl tRNA as initiator of the chain. Methionyl tRNA could preferentially bind to the acceptor site. Under these conditions, the formyl methionyl tRNA would occupy the donor site first and could therefore function as an initiator of the chain. The preferential binding of formyl methionyl tRNA to the donor site of the ribosomes using AUG as codon could result from a special configuration of the tRNA. 

Nomura, M. and Lowry, C. V. (1967) Phage f2 RNA-directed binding of formyl-methionyl-tRNA to ribosomes and the role of 30S ribosomal subunits in initiation of protein synthesis. Proc. Nat. Acad. Sci. U.S.A. 58, 946-953. 

Initiation in prokaryotes involves binding of mRNA by small ribosomal subunit (30S), followed by association of the fMet-tRNAmet (initiator formyl-methionyl-tRNAmel) that recognizes the initiation codon. Large ribosomal subunit (50S) then joins to form the 70S initiation complex. In eukaryotes, the initiator aminoacyl-tRNA is not formylated. Instead Met-tRNAf161 forms 40S preinitiation complex with small ribosomal subunit (40S) in the absence of mRNA. The association of mRNA results in a 40S preinitiation complex, which forms an 80S initiation complex after large ribosomal subunit (60S) joins. 

Both prokaryotes and eukaryotes initiate protein synthesis with a specialized methionyl-tRNA in response to an AUG initiation codon. Eukaryotes, however, use an initiator met—tRNAmeti—that is not formylated. Recognition of the initiator AUG is also different. Only one coding sequence exists per eukaryotic mRNA, and eukaryotic mRNAs are capped. Initiation, therefore, uses a specialized capbinding initiation factor to position the mRNA on the small riboso-mal subunit. Usually, the first AUG after the cap (that is, 3 to it) is used for initiation. 

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. 

Synthesis of fMet occurs on its tRNA. The tRNA is charged by MetRS with methionine, which then is formylated by methionyl-tRNA formyltransferase (MTF) in the presence of the formyl donor NlO-formyltetrahydrofolate (27). Initiation factor IF2 sequesters the fMet-tRNA and excludes it from the ribosomal A site. Instead, IF2-bound fMet-tRNA is transported to the ribosomal P site. EF-Tu GTP can bind methionyl-tRNA , although at a lower affinity than... 

Figure 29.21. Formylation of Methionyl-tRNA. Initiator tRNA (tRNAf) is first charged with methionine, and then a formyl group is transferred to the methionyl- tRNAf firomiV lo.formyltetrahydrofolate.

A. Methionine, the amino add that initiates the synthesis of proteins, is subsequently cleaved from the protein. The only codon for methionine is AUG, which serves as the codon for methionine residues within a protein as well as the initiating residue. The methionyl-tRNA for initiation is formylated in bacterial cells and in mitochondria. 

The initiator tRNA molecule in prokaryotes—tRNA — has several properties that distinguish it from all other tRNA molecules. One feature is that the tRNA is first acylated with methionine, and then the methionine is modified. Acylation is by methionyl tRNA synthetase, which also charges tRNA . However, the methionine of charged tRNA is immediately recognized by another enzyme, tRNA methionyl transformylase, which transfers a formyl group from N °-formyltetrahydrofolate (fTHF) to the amino group of the methionine to form... 

Methionine is linked to these two kinds of tRNAs by the same aminoacyl-iRNA synthetase. A specific enzyme then formylates the amino group of methionine attached to tRNAf (Figure 30.17), The activated formyl donor in this reaction is N hformyltetrahydrofolate (p. 689). It is significant that free methionine and methionyl-tRNA., are not substrates for this trans-formylase. 

Ribosomes needed for translation in the PURE system are isolated from E. coli using sucrose-density gradient centrifugation. The protein factors necessary for translation in E. coli are recombinantly expressed as His-tagged fusions, and purified to homogeneity. These include the factors for initiation (IFl, IF2, and IF3), elongation (EF-G, FF-Tu, FF-Ts), peptide chain release (RFl and RF3), ribosome recycling (RRF), methionyl-tRNA transformylase (MTF) for formylation of the initial Met-tRNA, and the 20 aminoacyl-tRNA synthetases (ARSs) for transfer RNA (tRNA) recy-... 

Essential modification reactions of aminoacyl-tRNAs. In bacteria the initiator tRNA needed to start the S5mthesis of a polypeptide is initially aminoacyl-ated by methionine, but the methionyl-tRNA must then be Af-formylated by transfer of a formyl group from Af °-formyltetrahydrofolate (Fig. 15-18 ... 

The initiation process differs for prokaryotes and eukaryotes (Table 15.3). In bacteria, the initiating methionyl-tRNA is formylated, producing a formyl-methionyl-tRNAf that participates in formation of the initiation complex (Fig. 15.9). Only three initiation factors (IFs) are required to generate this complex in prokaryotes, compared with the dozen or more required by eukaryotes. The ribosomes also differ in size. Prokaryotes have 70S ribosomes, composed of 308 and 50S subunits, and eukaryotes have SOS ribosomes, composed of 40S and 60S subunits. Unlike eukaryotic mRNA, bacterial mRNA is not capped. Identification of the initiating AUG triplet in prokaryotes occurs when a sequence in the mRNA (known as the Shine-Dalgamo sequence) binds to a complementary sequence near the 3 -end of the 16S rRNA of the small ribosomal subunit. 

During initiation of translation, the methionyl-tRNA species, tRNA is used exclusively. Presumably, the unique nucleotide sequence of this tRNA molecule is required for the initial interaction with the small ribo-somal subunit, the leader region of mRNA, initiation factors, and GTP, to give the first initiation complex. It is unlikely that formylation, which can only occur with methionyl- tRNA, is essential for initiation, as this is not the case in eukaryotes. Therefore, it appears that tRNA is excluded from initiation because essential structural requirements in the tRNA are not present. 

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. 

The first amino acid in the synthesis of all bacterial polypeptides is N-formylmethionine (fMet), i.e. a modified methionine residue with a formyl group attached to its amino group. Two types of methionyl-tRNA exist one binds formylmethionine and the other, normal methionine. Both have the same anticodon and recognize the AUG codon characteristic of methionine. However, only formylmethionyl-tRNA binds at the initiating AUG codon while normal methionyl-tRNA is bound at all internal AUG codons. 

The codon AUG, for methionine, serves as the initiation codon. The nucleotide sequence preceding AUG probably also plays a role in location of the start signal. As in prokaryotes, a special methionyl-tRNA (Met-tRNAf) functions as the initiator tRNA in eukaryotes. This tRNA is capable of being formylated by a bacterial enzyme (thus the subscript... 

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