TRNA initiator

In the following section, we describe protocols for tests aimed at screening for compounds capable of interfering with some of the main activities of this factor, such as (a) recognition and binding of initiator tRNA (b) codon-dependent ribosomal binding of fMet-tRNA leading to the formation of a 30S or 70S initiation complex (c) ribosome-dependent hydrolysis of GTP and (d) accommodation of fMet-tRNA in the ribosomal P-site and formation of the first peptide bond (initiation dipeptide formation). 

The activity of IF2 in binding fMet-tRNA was measured quantifying the protection conferred by these proteins on the initiator tRNA with respect to spontaneous hydrolysis occurring at alkaline pH (Gualerzi et al., 1991 Petersen et al., 1979). Reaction mixtures (50 pi) in Buffer F contained 22 pM f 35S]Met-tRNA, an appropriate amount of protein that is capable of protecting approximately 80% of the initiator tRNA after 60 min incubation as well as increasing concentrations of the antibiotic to be tested. Samples (20 pi), withdrawn after 0 and 60 min of incubation at 37°, are spotted on Whatman 3MM paper discs for determination of the acid-insoluble radioactivity by the cold TCA procedure, described previously. 

Pestova, T. V., and Hellen, C. U. (2003). Translation elongation after assembly of ribosomes on the Cricket paralysis virus internal ribosomal entry site without initiation factors or initiator tRNA. Genes Dev. 17, 181—186. 

Hradec J, Spiegelhalder B, Preussmann R. 1988. The initiator tRNA acceptance assay as a shortterm test for carcinogens. 2. Results with ten compounds selected by the International Programme on Chemical Safety for the evaluation of short-term tests for carcinogens. Carcinogenesis 9 843-846. 

After attachment of amino acids to tRNA, the amino acids are assembled beginning with the amino terminus and proceeding in the direction of the carboxy terminus. The ribosome is the machinery that translates the mRNA into protein. The ribosome is a very complex protein that contains ribosomal RNA as a functional and structural component. The ribosome assembles around the mRNA, and the cap and other signals allow alignment of the mRNA into the correct position. The initial assembly of the mRNA into the ribosome requires association of the small ribosomal subunit with an initiator tRNA (Met or fMet). Small is a misstatement, because the small ribosomal subunit is a large, complex assembly of numerous smaller proteins—it s just smaller than the... 

The charged initiator tRNA becomes bound to the AUG start codon on the messt e through base pairing with its anticodon. The initiator tRNA in prokaryotes carries fmet, whereas the initiator tRNA in eukaryotes carries Met. 

Answer C. eIF-2 designates a protein factor of the initiation phase in eukaryotic translation. The only event listed that would occur during this phase is placement of initiator tRNA in the P-site. 

The fimction of eIF-2 is illustrated schematically in Fig. 1.55. eIF-2 belongs to the superfamily of regulatory GTPases (see ch. 5). elF-2 fulfills the task of bringing the methionyl-initiator-tRNA to the 40S subimit of the ribosome. The active eIF-2 GTP form binds the methionyl-initiator-tRNA, associates with the cap structure of the mRNA, then commences to scan along the mRNA. Once an AUG codon is encoimte-red, the boimd GTP is hydrolyzed to GDP, resulting in the dissociation of the... 

Fig. 1. 55. The function of eIF-2 in eucaryotic translation. eIF-2, the initiator protein for the translation is a regulatory GTPase that occurs in an active GTP-form and in an inactive GDP form (see ch. 5). The active eIF-2 GTP forms a complex with the initiator-tRNA, fMet-tRNA "" and the 40S subunit of the ribosome. This complex binds to the cap structure of mRNA to initiate the scanning process. eIF-2 undergoes an activation cycle typical for regulatory GTPases the inactive eIF-2 GDP fom is activated with the assistance of the eIF-2B protein into the active elF-2 GTP form. eIF-2B acts as a G-nucleotide exchange factor in the cycle (see ch. 5).

Translational regulation has been particularly well studied in reticulocytes. One such mechanism in these cells involves eIF2, the initiation factor that binds to the initiator tRNA and conveys it to the ribosome when Met-tRNA has bound to the P site, the factor eIF2B... 

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... 

Figure 29-7 (A) Generalized cloverleaf diagram of all tRNA sequences except for initiator tRNAs numbered as in yeast tRNAae (Fig. 5-30). Invariant bases A, C, G, T, U, and semivariant bases Y (pyrimidine base), R (purine base), H (hypermodified purine base). The dotted regions (a, P, variable loop) contain different numbers of nucleotides in various tRNA sequences. See Rich.179 (B) L form of the yeast phenyl-alanine-specific tRNAphe. The structure is the same as that in Fig. 5-31 but has recently been redetermined at a resolution of 0.20 nm.175 The new data revealed the presence of ten bound Mg2+ ions (green circles) as well as bound spermine (green).

Initiator tRNAs. While the T /C sequence has been found in all bacterial and most eukaryotic tRNAs examined, it is replaced by UCG in eukaryotic initiator tRNAs. In these tRNAs the preceding two nucleotides, beginning in the stem of loop IV, are also conserved the complete conserved sequence being GAUCG.184 Other characteristics of initiator tRNAs are the absence of base-pairing between residues 1, and 72, and the presence of C rather than G at position 1, A rather than G at position 72, and CCU in place of the two dihydroU residues in loop I.185 Initiator tRNAs of chloroplasts resemble those of bacteria,186 whereas archaeobacteria have their own unique peculiarities.187 These include the presence of a hypermod-ified base known as archaeosine (p. 1456) in position 15 of the dihydroU loop.188 189... 

Discrimination between some pairs of tRNAs depends entirely on the anticodon sequence. For example, tRNAMet contains the anticodon CAU. That for a minor tRNAIle is the same except that the cytosine has been posttranscriptionally modified by covalent linkage of a molecule of lysine via its e-amino group to C2 of the cytosine. The latter base (Iysidine) is correctly recognized by E. coli isoleucyl-tRNA synthetase but, if the cytosine is unmodified, it is aminoacylated by methionyl-tRNA synthetase.192 In most instances the acceptor specificity, or tRNA identity, is not determined solely by the anticodon sequence. Thus, when a methionine initiator tRNA was modified to contain a tryptophan anticodon, it was only partially charged with tryptophan in vivo. However, when A73 of the methionine tRNA was also converted to G73, only tryptophan was inserted.193 Nucleotide 73 (Fig. 

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 ... 

The translation of every protein begins with the incorporation of the amino acid methionine. A unique initiator tRNA, tRNAjMet, is responsible for the incorporation of this initiating methionine in all protein-synthesizing systems, and it also plays an important role in selecting the appropriate translation start site in mRNA. Generally, only two tRNAs occur in cells that specify methionine. We designate the one that is responsible for the incorporation of internal methio-... 

Even though specific differences distinguish the initiation process in eukaryotes and prokaryotes, three things must be accomplished to initiate protein synthesis in all systems (1) The small ribosomal subunit must bind the initiator tRNA (2) the appropriate initiating codon on mRNA must be located and (3) the large ribosomal subunit must associate with the complex of the small subunit, the initiating tRNA, and mRNA. Nonribosomal proteins, known as initiation factors (IFs), participate in each of these three processes. IFs interact transiently with a ribosome during initiation and thus differ from ribosomal proteins, which remain continuously associated with the same ribosome. 

E. coli has three initiation factors (fig. 29.13) bound to a small pool of 30S ribosomal subunits. One of these factors, IF-3, serves to hold the 30S and 50S subunits apart after termination of a previous round of protein synthesis. The other two factors IF-1 and IF-2, promote the binding of both fMet-tRNA,Mel and mRNA to the 30S subunit. As we noted before, the binding of mRNA occurs so that its Shine-Dalgarno sequence pairs with 16S RNA and the initiating AUG sequence with the anticodon of the initiator tRNA. The 30S subunit and its associated factors can bind fMet-tRNAjMet and mRNA in either order. Once these ligands are found, IF-3 dissociates from the 30S, permitting the 50S to join the complex. This releases the remaining initiation factors and hydrolyzes the GTP that is bound to IF-2. The initiation step in prokaryotes requires the hydrolysis of one equivalent of GTP to GDP and Pj. 

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... 

As in prokaryotes, a special initiator tRNA is required for initiation and is distinct from the tRNA that recognizes and binds to codons for methionine at internal positions in the mRNA. When charged with methionine ready to begin initiation, this is known as Met-tRNAimet. 

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... 

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