Structural analysis results in a detailed mechanism for the hydrolysis of the mischarged tRNA. The two analogs represent the substrate for editing at the activation step (pretransfer editing) and after completion of the amino acid transfer on the tRNA (posttransfer editing). Four structures were solved, comprising the apo form and three complexes with either serine or nonhydrolyzable analogs of seryl-adenylate and of seryl-3 Ј -adenosine, which is similar to the extremity of a mischarged tRNA. In order to understand the mechanism of this highly specific reaction, we have produced and crystal- lized the N-terminal part of ThrRS comprising the editing domain (residues 1–224). the erro- neously bound serine and thus ensures faithful aminoacylation. The residues discussed in the text are indicated by stars (numbering of E. The alignment was performed with the PipeAlign package ( Black shading indicates residues conserved in more than 90% of the sequences of the group. Bottom, framed in green: AlaRS domain equivalent to N2, found in the C-terminal part of the enzyme, represented by sequences from E. coli sequence (this work), the eukaryal Streptomyces coelicolor and Homo sapiens, and the archaeal Thermoplasma volcanium (Aa). Top, framed in orange: the archaeal editing domain, found in the 14 sequences of groups (Ab) and (Ad), represented by the sequences from Pyrococcus abyssi, Methanosarcina acetivorans, and Sulfolobus solfataricus middle, framed in red: the N2 domain in eukaryotic, eubacterial, and archaeal ThrRSs, represented by the bacterial E. (B) Sampling of sequences after alignment of the N2 or equivalent domain, in three groups of proteins. The brackets stand for domain N1 and additional N-terminal extension, as found in higher eukaryotes (b) archaeal ThrRSs, with a different editing domain (vertical orange stripes, 11 sequences) (c) ThrRSs from Mycobacterium, Buchnera, or mitochondria, which have no N-terminal domain, or domains bearing only very weak homology with the N2 domain (10 sequences) (d) Archaebacteria S. (a) eukaryotic, eubacterial, and archaeal ThrRSs containing the N2 domain, striped red (103 out of 130 sequences). Sequence Analysis (A) Schematic representation of the domain organization of ThrRSs, with the catalytic (central) domain in yellow (the black bars represent the conserved motifs 1,2,3) and the anticodon binding domain in purple.
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