Functional analysis of peptide motif for RNA microhelix binding suggests new family of RNA-binding domains

Pouplana, Lluı́s Ribas de; Buechter, Douglas D.; Sardesai, Niranjan Y.; Schimmel, Paul

doi:10.1093/emboj/17.18.5449

Cited by 19 publications

(24 citation statements)

References 61 publications

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“…Altogether, the results of the steady-state and single-turnover kinetic analyses suggest that the hDRS extension likely interacts with tRNA or the active site in a manner that may provide additional understanding of the process of synthetase⅐RNA interactions. Furthermore, in contrast to the lysine-rich extensions described in several recent reports (10,26,27), the 32-residue extension in hDRS is short and is not highly basic. In this paper, we focus on the tRNA binding by the amino-terminal extension in hDRS using fluorescence spectroscopy and circular dichroism (CD).…”

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confidence: 69%

Magnesium Ion-mediated Binding to tRNA by an Amino-terminal Peptide of a Class II tRNA Synthetase

Hammamieh

Yang

2001

Journal of Biological Chemistry

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Aspartyl-tRNA synthetase is a class II tRNA synthetase and occurs in a multisynthetase complex in mammalian cells. Human Asp-tRNA synthetase contains a short 32-residue amino-terminal extension that can control the release of charged tRNA and its direct transfer to elongation factor 1␣; however, whether the extension binds to tRNA directly or interacts with the synthetase active site is not known. Full-length human AspRS, but not amino-terminal 32 residue-deleted, fully active AspRS, was found to bind to noncognate tRNA fMet in the presence of Mg 2؉ . Synthetic amino-terminal peptides bound similarly to tRNA fMet , whereas little or no binding of polynucleotides, poly(dA-dT), or polyphosphate to the peptides was found. The apparent binding constants to tRNA by the peptide increased with increasing concentrations of Mg 2؉ , suggesting Mg 2؉ mediates the binding as a new mode of RNA⅐peptide interactions. The binding of tRNA fMet to amino-terminal peptides was also observed using fluorescence-labeled tRNAs and circular dichroism. These results suggest that a small peptide can bind to tRNA selectively and that evolution of class II tRNA synthetases may involve structural changes of amino-terminal extensions for enhanced selective binding of tRNA.Aminoacyl-tRNA synthetases catalyze the covalent attachments of amino acids to cognate tRNAs in the first step of protein biosynthesis. Extensive studies of the structure and function of this family of enzymes have provided excellent understanding of fundamental principles of RNA-protein interactions and structures of synthetases. Almost all synthetases contain a core catalytic domain carrying out adenylation of an amino acid and an anti-codon binding domain essential for aminoacylation of cognate tRNA. The core catalytic domains in class I synthetases resemble dinucleotide binding folds and reside in the amino termini, whereas a seven-stranded antiparallel sheet with three ␣-helices characterizes active sites in class II synthetases and locates in the carboxyl termini of synthetases.Beyond the basic amino-and carboxyl-terminal catalytic domains, eukaryotic and mammalian synthetases (1-3) have evolved idiosyncratic extensions dispensable for aminoacylation of tRNA. Additionally, extensive association of synthetases occurs in high eukaryotic organisms; thus, 9 of the 20 synthetases associate as a multienzyme complex, ProRS and GluRS form a fused GluProRS (4, 5), and ValRS associates with the EF1H as a separate complex (6, 7). The function of the extensions in synthetases has attracted more attention recently. Controlled proteolysis or hydrophobic interaction chromatography dissociates several synthetases from the synthetase complex without significantly affecting enzymatic activities (8, 9); thus, the extensions in mammalian synthetases likely play pivotal roles in the structural organization of the synthetase complex. Extensions in synthetases can be involved in RNA binding (10, 11) or function as cytokines (12)(13)(14). It appears that extensions in synthetases are multifun...

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confidence: 69%

Magnesium Ion-mediated Binding to tRNA by an Amino-terminal Peptide of a Class II tRNA Synthetase

Hammamieh

Yang

2001

Journal of Biological Chemistry

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“…The A76 residues in the first and second models are highlighted in yellow and blue, respectively. Amino acid residues important for the aminoacylation or editing activity (17,28,38,39,45,46) are shown as stick models. Ala-SA in the aminoacylation site and the editing-site zinc ion are depicted as cpk models.…”

Section: Position Of the Editing Domain The Editing Domain Of A Fulmentioning

confidence: 99%

“…Therefore, the bacterial AlaRSs might have the editing domain in a different location from that in the archaeal AlaRS, relative to the aminoacylation/tRNA-recognition domains, thus allowing the tRNA to shift easily between the 2 active sites. In E. coli AlaRS, the aminoacylation/tRNA-recognition domains and the editing domain are both able to recognize the G3⅐U70 base pair in the tRNA acceptor stem (17), involving Arg-314 on the tRNArecognition domain and Arg-693 on the editing domain of E. coli AlaRS (17,39). Intriguingly, in the present A. fulgidus AlaRS structure, Arg-371 (Mid1) and Arg-731, which correspond to the G3⅐U70 recognition residues Arg-314 and Arg-693, respectively, are close to the putative tRNA binding site in the alternative groove (Fig.…”

Section: Position Of the Editing Domain The Editing Domain Of A Fulmentioning

confidence: 99%

Unique protein architecture of alanyl-tRNA synthetase for aminoacylation, editing, and dimerization

Naganuma

Sekine

Fukunaga

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Alanyl-tRNA synthetase (AlaRS) specifically recognizes the major identity determinant, the G3:U70 base pair, in the acceptor stem of tRNA Ala by both the tRNA-recognition and editing domains. In this study, we solved the crystal structures of 2 halves of Archaeoglobus fulgidus AlaRS: AlaRS-⌬C, comprising the aminoacylation, tRNA-recognition, and editing domains, and AlaRS-C, comprising the dimerization domain. The aminoacylation/tRNA-recognition domains contain an insertion incompatible with the class-specific tRNA-binding mode. The editing domain is fixed tightly via hydrophobic interactions to the aminoacylation/tRNA-recognition domains, on the side opposite from that in threonyl-tRNA synthetase. A groove formed between the aminoacylation/tRNA-recognition domains and the editing domain appears to be an alternative tRNA-binding site, which might be used for the aminoacylation and/or editing reactions. Actually, the amino acid residues required for the G3:U70 recognition are mapped in this groove. The dimerization domain consists of helical and globular subdomains. The helical subdomain mediates dimerization by forming a helixloop-helix zipper. The globular subdomain, which is important for the aminoacylation and editing activities, has a positively-charged face suitable for tRNA binding.crystal structure ͉ dimerization domain ͉ aminoacyl-tRNA synthetase ͉ proofreading ͉ wobble base pair

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“…The specificity for G:U versus U:G at the 3:70 position of microhelix Ala was reduced Ϸ5-fold for 368N-MF␤25 relative to the free MF␤2 peptide. This reduction probably reflects the serendipitous weak nonspecific RNA-binding determinants in fragment 368N that affect the context for RNA binding by the fusion peptide (35). Further selections could be placed on the fusion proteins to ameliorate these effects.…”

Section: Mutational Analysis Of Fusion Peptidementioning

confidence: 99%

RNA recognition by designed peptide fusion creates "artificial" tRNA synthetase

Frugier¹,

Giegé²,

Schimmel³

2003

Proc. Natl. Acad. Sci. U.S.A.

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The genetic code was established through aminoacylations of RNA substrates that emerged as tRNAs. The 20 aminoacyl-tRNA synthetases (one for each amino acid) are ancient proteins, the active-site domain of which catalyzes formation of an aminoacyl adenylate that subsequently reacts with the 3 end of bound tRNA. Binding of tRNA depends on idiosyncratic (to the particular synthetase) domains and motifs that are fused to or inserted into the conserved active-site domain. Here we take the domain for synthesis of alanyl adenylate and fuse it to ''artificial'' peptide sequences (28 aa) that were shown previously to bind to the acceptor arm of tRNA Ala . Certain fusions confer aminoacylation activity on tRNA Ala and on hairpin microhelices modeled after its acceptor stem. Aminoacylation was sensitive to the presence of a specific G:U base pair known to be a major determinant of tRNA Ala identity. Aminoacylation efficiency and specificity also depended on the specific peptide sequence. The results demonstrate that barriers to RNA-specific aminoacylations are low and can be achieved by relatively simple peptide fusions. They also suggest a paradigm for rationally designed specific aminoacylations based on peptide fusions.

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Functional analysis of peptide motif for RNA microhelix binding suggests new family of RNA-binding domains

Cited by 19 publications

References 61 publications

Magnesium Ion-mediated Binding to tRNA by an Amino-terminal Peptide of a Class II tRNA Synthetase

Magnesium Ion-mediated Binding to tRNA by an Amino-terminal Peptide of a Class II tRNA Synthetase

Unique protein architecture of alanyl-tRNA synthetase for aminoacylation, editing, and dimerization

RNA recognition by designed peptide fusion creates "artificial" tRNA synthetase

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