A study of the interaction of Escherichia coli initiation factor IF2 with formylmethionyl‐tRNAMetf by partial digestion with cobra venom ribonuclease

The conserved positions of the eukaryotic cytoplasmic initiator tRNA have been suggested to be important for the initiation of protein synthesis. However, the role of these positions is not known. We describe in this report a functional analysis of the yeast initiator methionine tRNA (tRNAVet), using a novel in vivo assay system which is not dependent on suppressor tRNAs. Strains of Saccharomyces cerevisiae with null alleles of the four initiator methionine tRNA (IMT) genes were constructed. Consequently, growth of these strains was dependent on tRNAe et encoded from a plasmid-derived gene. We used these strains to investigate the significance of the conserved nucleosides of yeast tRNA,Met in vivo. Nucleotide substitutions corresponding to the nucleosides of the yeast elongator methionine tRNA (tRNAmet) have been made at all conserved positions to identify the positions that are important for tRNA,4et to function in the initiation process. Surprisingly, nucleoside changes in base pairs 3-70, 12-23, 31-39, and 29-41, as well as expanding loop I by inserting an A at position 17 (A17) had no effect on the tester strain. Nucleotide substitutions in positions 54 and 60 to cytidines and guanosines (C54, G54, C60, and G60) did not prevent cell growth. In contrast, the double mutation U/rT54C60 blocked cell growth, and changing the A-U base pair 1-72 to a G-C base pair was deleterious to the cell, although these tRNAs were synthesized and accepted methionine in vitro. From our data, we suggest that an A-U base pair in position 1-72 is important for tRNA,eet function, that the hypothetical requirement for adenosines at positions 54 and 60 is invalid, and that a U/rT at position 54 is an antideterminant distinguishing an elongator from an initiator tRNA in the initiation of translation.In the translational decoding of mRNA, codons are read by a unique set of tRNA species. Each isoacceptor is usually encoded by more than one copy of the gene. To study the function of a tRNA in protein synthesis, a common strategy is to generate tRNA mutations. To date, phenotypic changes in vivo have been scored either by different types of suppressor tRNAs (nonsense, missense, and frameshift, i.e., a tRNA with an already existing mutation) or by an in vivo system in which the rate of aminoacyl-tRNA selection is measured in competition with a natural tRNA'U frameshifter (14,19,39). The former strategy has some limitations. First, the anticodon is a major identity element for some tRNA synthetases (41, 49), including the methionyl-tRNA synthetase from Escherichia coli (50). Therefore, the translational efficiency of the suppressor tRNA cannot be distinguished from its capacity to be aminoacylated. Second, in the translational process, a stop codon is a signal for the release factors to stop translation. Thus, the translational efficiency of the suppressor tRNA will be masked, as the tRNA must compete with release factors for the stop codon. In this report, an in vivo system which overcomes these obstacles by being dependent on one single I...

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“…interact with this surface (20,24,42,51,57,58,60). We can only hypothesize what the recognition/restriction elements of these factors might be.…”

Section: Resultsmentioning

confidence: 99%

Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae.

Pawel‐Rammingen¹,

Åström²,

Byström³

1992

Mol. Cell. Biol.

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“…The experiments indicate binding of IF2 to the acceptor stem, position 12 to 13 in the D-stem, two sites in the anticodon stem, and parts of the T-loop and the minor groove of the fMet-tRNA f Met T-stem ( Fig. 6) (166,243). Based on a similar cleavage pattern of RNase VI in the anticodon stem of Met-tRNA f Met bound to MTF, it was proposed that the interaction between fMettRNA f Met and IF2 induces a conformational change in the anticodon stem (122).…”

Section: Initiator Trnamentioning

confidence: 96%

Initiation of Protein Synthesis in Bacteria

Laursen

Sørensen

Mortensen

et al. 2005

Microbiol Mol Biol Rev

530

506

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Valuable information on translation initiation is available from biochemical data and recently solved structures. We present a detailed description of current knowledge about the structure, function, and interactions of the individual components involved in bacterial translation initiation. The first section describes the ribosomal features relevant to the initiation process. Subsequent sections describe the structure, function, and interactions of the mRNA, the initiator tRNA, and the initiation factors IF1, IF2, and IF3. Finally, we provide an overview of mechanisms of regulation of the translation initiation event. Translation occurs on ribonucleoprotein complexes called ribosomes. The ribosome is composed of a large subunit and a small subunit that hold the activities of peptidyltransfer and decode the triplet code of the mRNA, respectively. Translation initiation is promoted by IF1, IF2, and IF3, which mediate base pairing of the initiator tRNA anticodon to the mRNA initiation codon located in the ribosomal P-site. The mechanism of translation initiation differs for canonical and leaderless mRNAs, since the latter is dependent on the relative level of the initiation factors. Regulation of translation occurs primarily in the initiation phase. Secondary structures at the mRNA ribosomal binding site (RBS) inhibit translation initiation. The accessibility of the RBS is regulated by temperature and binding of small metabolites, proteins, or antisense RNAs. The future challenge is to obtain atomic-resolution structures of complete initiation complexes in order to understand the mechanism of translation initiation in molecular detail

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“…It may be speculated that IF2 and methionyl-tRNA synthetase interact with the anticodon stem region of the initiator tRNA in a similar manner, since they each have a similarly structured subdomain and both interact with the initiator tRNA. Moreover, footprinting studies show that IF2 and methionyl-tRNA synthetase protect the same position in the anticodon stem of the initiator tRNA against RNase cleavage (37,38). The SCfold domain in methionyl-tRNA synthetase is followed by a helix bundle that interacts with the anticodon stem and loop of the tRNA (39).…”

Section: Discussionmentioning

confidence: 99%

A Conserved Structural Motif at the N Terminus of Bacterial Translation Initiation Factor IF2

Laursen

Mortensen

Sperling‐Petersen

et al. 2003

Journal of Biological Chemistry

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The 18-kDa Domain I from the N-terminal region of translation initiation factor IF2 from Escherichia coli was expressed, purified, and structurally characterized using multidimensional NMR methods. Residues 2-50 were found to form a compact subdomain containing three short ␤-strands and three ␣-helices, folded to form a ␤␣␣␤␤␣ motif with the three helices packed on the same side of a small twisted ␤-sheet. The hydrophobic amino acids in the core of the subdomain are conserved in a wide range of species, indicating that a similarly structured motif is present at the N terminus of IF2 in many of the bacteria. External to the compact 50-amino acid subdomain, residues 51-97 are less conserved and do not appear to form a regular structure, whereas residues 98 -157 form a helix containing a repetitive sequence of mostly hydrophilic amino acids. Nitrogen-15 relaxation rate measurements provide evidence that the first 50 residues form a well ordered subdomain, whereas other regions of Domain I are significantly more mobile. The compact subdomain at the N terminus of IF2 shows structural homology to the tRNA anticodon stem contact fold domains of the methionyl-tRNA and glutaminyl-tRNA synthetases, and a similar fold is also found in the B5 domain of the phenylalanine-tRNA synthetase. The results of the present work will provide guidance for the design of future experiments directed toward understanding the functional roles of this widely conserved structural domain within IF2.

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A study of the interaction of Escherichia coli initiation factor IF2 with formylmethionyl‐tRNA^Met_f by partial digestion with cobra venom ribonuclease

Cited by 17 publications

References 8 publications

Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae.

Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae.

Initiation of Protein Synthesis in Bacteria

A Conserved Structural Motif at the N Terminus of Bacterial Translation Initiation Factor IF2

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