Connectivity-based ab initio phasing at different solvent levels.

Andrei Fokine; Natalia Lunina; Vladimir Lunin; Alexandre Urzhumtsev

doi:10.1107/s0907444903004359

Home/Archive/PubMed

PubMed · 12777801

Connectivity-based ab initio phasing at different solvent levels.

Andrei Fokine·Natalia Lunina·Vladimir Lunin·Alexandre Urzhumtsev

View original source ↗Export metadata ↓

Abstract

The connectivity-based phasing method has been applied independently to three neutron diffraction data sets obtained from the same crystal of tRNA(Asp)-aspartyl-tRNA synthetase complex but at different concentrations of D(2)O/H(2)O, thus masking different components of the crystal. The obtained low-resolution images correlate perfectly with the solvent level.

Explore related subjects

Aspartate-tRNA Ligase Deuterium Oxide Models, Molecular Neutron Diffraction Nucleic Acid Conformation Protein Conformation RNA, Transfer, Asp Solvents Statistics as Topic Water Journal Article Research Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Andrei Fokine, Natalia Lunina, Vladimir Lunin, Alexandre Urzhumtsev. 2003-04-25. Connectivity-based ab initio phasing at different solvent levels.. https://doi.org/10.1107/s0907444903004359

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2006

A non-discriminating aspartyl-tRNA synthetase from Halobacterium salinarum.

The tRNA-dependent transamidation pathway is the essential route for Asn-tRNA(Asn) formation in organisms that lack an asparaginyl-tRNA synthetase. This pathway relies on a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS encoded by aspS), an enzyme with relaxed tRNA specificity, to form Asp-tRNA(Asn). The misacylated tRNA is then converted to Asn-tRNA(Asn) by the action of an Asp-tRNA(Asn) amidotransferase. Here we show that Asn-tRNA(Asn) formation in the extreme halophile Halobacterium salinarum also occurs by this transamidation mechanism, and we explore the property of the haloarchaeal AspRS to aspartylate tRNA(Asn) in vivo and in vitro. Transformation of the E. coli trpA34 strain with the H. salinarum aspS and tRNA(Asn) genes led to restoration of tryptophan prototrophy by missense suppression of the trpA34 mutant with heterologously in vivo formed Asp-tRNA(Asn). The haloarchaeal AspRS works well at low and high (0.1-3 M) salt concentrations but it is unable to use Escherichia coli tRNA as substrate. We show that mutations of two amino acids (H26 and P84) located in the AspRS anticodon binding domain limit the specificity of this nondiscriminating enzyme towards tRNA(Asn). Thus, as was observed in an archaeal discriminating AspRS and a bacterial ND-AspRS, amino acids in these positions influence the enzyme's tRNA selection.

Aspartate-tRNA Ligase↗

PubMed2005

A residue-pairwise generalized born scheme suitable for protein design calculations.

We describe an efficient generalized Born (GB) approximation for proteins, in which the interaction energy between two amino acids depends on the whole protein structure, but can be accurately computed from residue-pairwise information. Two results make the scheme pairwise. First, an accurate expression exists for the interaction energy between two residues R and R' that depends on the product B = BRBR' of their residue Born solvation radii. Second, this expression is accurately fitted by a parabolic function of B; the (three) fitting coefficients depend only on the pair RR', not on its environment. In effect, the quantity B captures all the information that is relevant about the pair's dielectric environment. The method is tested with calculations on several hundred structures of the proteins trpcage, BPTI, ubiqutin, and thoredoxin. It yields solvation energies in better agreement with Poisson calculations than a traditional GB formulation. We also compute the effect of the protein/solvent environment on the interactions between pairs of charged residues in the active site of the enzyme aspartyl-tRNA synthetase. Our method captures this effect as accurately as traditional GB. Because it is residue-pairwise, the method can be incorporated into efficient protocols for rotamer placement and computational protein design.

Aspartate-tRNA Ligase↗

PubMed2005

An intricate RNA structure with two tRNA-derived motifs directs complex formation between yeast aspartyl-tRNA synthetase and its mRNA.

Accurate translation of genetic information necessitates the tuned expression of a large group of genes. Amongst them, controlled expression of the enzymes catalyzing the aminoacylation of tRNAs, the aminoacyl-tRNA synthetases, is essential to insure translational fidelity. In the yeast Saccharomyces cerevisiae, expression of aspartyl-tRNA synthetase (AspRS) is regulated in a process necessitating recognition of the 5' extremity of AspRS messenger RNA (mRNA(AspRS)) by its translation product and adaptation to the cellular tRNA(Asp) concentration. Here, we have established the folding of the approximately 300 nucleotides long 5' end of mRNA(AspRS) and identified the structural signals involved in the regulation process. We show that the regulatory region in mRNA(AspRS) folds in two independent and symmetrically structured domains spaced by two single-stranded connectors. Domain I displays a tRNA(Asp) anticodon-like stem-loop structure with mimics of the aspartate identity determinants, that is restricted in domain II to a short double-stranded helix. The overall mRNA structure, based on enzymatic and chemical probing, supports a three-dimensional model where each monomer of yeast AspRS binds one individual domain and recognizes the mRNA structure as it recognizes its cognate tRNA(Asp). Sequence comparison of yeast genomes shows that the features within the mRNA recognized by AspRS are conserved in different Saccharomyces species. In the recognition process, the N-terminal extension of each AspRS subunit plays a crucial role in anchoring the tRNA-like motifs of the mRNA on the synthetase.

Aspartate-tRNA Ligase↗