The 1.2 Å crystal structure of an E. coli tRNASer acceptor stem microhelix reveals two magnesium binding sites

https://doi.org/10.1016/j.bbrc.2009.06.048Get rights and content

Abstract

tRNA identity elements assure the correct aminoacylation of tRNAs by the cognate aminoacyl-tRNA synthetases. tRNASer belongs to the so-called class II system, in which the identity elements are rather simple and are mostly located in the acceptor stem region, in contrast to ‘class I’, where tRNA determinants are more complex and are located within different regions of the tRNA.

The structure of an Escherichia coli tRNASer acceptor stem microhelix was solved by high resolution X-ray structure analysis. The RNA crystallizes in the space group C2, with one molecule per asymmetric unit and with the cell constants a = 35.79, b = 39.13, c = 31.37 Å, and β = 111.1°. A defined hydration pattern of 97 water molecules surrounds the tRNASer acceptor stem microhelix. Additionally, two magnesium binding sites were detected in the tRNASer aminoacyl stem.

Introduction

The translation of the genetic code into proteins is assured by the ribosomal protein biosynthesis. A crucial step which assures the accuracy of the process is the correct aminoacylation of tRNAs with their cognate amino acids by the specific aminoacyl-tRNA synthetases. The tRNA identity is assured by tRNA determinants, which are sequence or structure motifs that determine the tRNA specificity.

There exist two classes of tRNA/aminoacyl-tRNA systems, class I and class II [1]. In class I, the tRNA identity elements are relatively complex and consist of sequence/structure motifs that are located in different regions of the tRNAs mostly including the anticodon. In class II, the identity elements are more simple and mostly located in the region of the aminoacyl stem, often including the discriminator base at position 73 [1]. The tRNA determinants in class II can be so simple as to consist of only one base pair [2].

The tRNASer/seryl-tRNA synthetase (SerRS) system belongs to the class II system and is well investigated. The minimal tRNASer consensus elements which assure the specificity consist of the base pairs A3-U70, G2-C71, the nucleotide C72, the discriminator base G73, and additionally C11-G24 in the D-stem [3], [4], [5], [6]. Also, interactions between the 3′-end of tRNASer and the SerRS could be shown by footprinting experiments [7]. The positions which contact the SerRS include the nucleotides C67, U68, C69, and U70, with additional contacts in the T-region of the tRNASer. The presence of the long extra arm in tRNASer is one of the main identity elements. This region shows backbone contacts to a conserved domain in the SerRS [4], [5]. This has been also shown for an archaebacterial seryl-tRNA synthetase [8], [9], in which the long extra arm has been described as a main determinant for tRNASer–synthetase interaction.

The 2.9 and 2.7 Å X-ray structures of the tRNASer complexed to Thermus thermophilus SerRS present the interaction of this specific tRNASer/SerRS system [10], [11]. The interaction occurs between the inside of the L-shaped tertiary tRNA structure and the protein. Contacts include the 5′- and 3′-ends of the tRNASer aminoacyl stem, the GCCA-end and the long, unique extra arm. Nucleotides in the aminoacyl region that are involved in the interaction, like G2-C71, C69, U68, contact amino acids from the loop 2 region of the seryl-tRNA synthetase. The authors point out that there are rather few base-specific interactions between the acceptor stem and the synthetase, and that the interactions are mainly presented by phosphate backbone contacts, which is in consistence with the footprinting experiments [7].

The structures of the complexes revealed a remarkable conformational change in the loop 2 region of the synthetase, representing either the state of the tRNA-binding form or of the seryl-AMP-binding form. These two conformations of the synthetase are described as T- or A-conformation [11]. Nevertheless, due to a partial incompleteness of the electron density map in the structure within the end of the aminoacyl stem and the 3′-CCA end of tRNASer, the specific local helical parameters of this part cannot be interpreted. Thus, we decided to investigate high resolution X-ray structures of tRNASer microhelices to complete the information including the specific local geometric parameters of the acceptor stems and to undertake superposition experiments to the synthetase [12], [13].

Via initial superposition calculations between a previously published 1.8 Å tRNASer microhelix structure [13] and the SerRS [10], [11], we could calculate a hypothetical interface, in which the following nucleotides were identified to interact with the synthetase: U66, C67, U68, C69, and U70 from the 3′-strand as well as G1 from the 5′-strand of the tRNASer acceptor stem helix [12]. The surface revealed the amino acids Gly 263, Phe 262, Ser 261, and Arg 267 of SerRS to be involved in the interaction to the tRNASer, which is in agreement with the structure of the complex [10], [11].

We focus on high resolution structure analysis of additional tRNASer microhelix isoacceptors, which differ in sequence, to further investigate the tRNASer/SerRS interactions. By the high resolution structures of the microhelices, the detailed local geometric parameters of the RNA, the role of single water molecules and possible metal ion binding sites can be analyzed in detail.

Here, we report the crystallization and X-ray structure determination of an Escherichia coli tRNASer acceptor stem microhelix at 1.2 Å resolution. The RNA crystallizes in the monoclinic space group C2 with the cell constants a = 35.79, b = 39.13, c = 31.37 Å and β = 111.1° and one molecule per asymmetric unit. The RNA is surrounded by a hydration pattern of 97 solvent molecules. Additionally, two magnesium binding sites could be detected within the tRNASer acceptor stem microhelix.

Section snippets

Materials and methods

Crystallization of the E. coli tRNASer acceptor stem microhelix. The sequence of the tRNASer microhelix was derived from the tRNA data base [14]. The two RNA 7-mer oligonucleotides with the sequences 5′-G1G2U3G4A5G6G7-3′ and 5′-C66C67U68C69A70C71C72-3′ were purchased from IBA (Göttingen, Germany) with HPLC-purification grade. The concentration of the single RNA strands was determined by alkaline hydrolysis considering the molar extinction coefficients of the single nucleotides and following

Crystallization and structure determination of the tRNASer acceptor stem microhelix

The E. coli tRNASer microhelix (Fig. 1, Fig. 2) was crystallized in 10% (v/v) MPD, 40 mM sodium cacodylate, pH 5.5, 20 mM cobalt hexamine, 80 mM sodium chloride, 20 mM magnesium chloride with equilibration against 35% (v/v) MPD at 294 K. Regularly crystals appeared after several days with the space group C2 and the cell constants constants: a = 35.79, b = 39.13, c = 31.37 Å and β = 111.1°. The crystals contained one RNA molecule per asymmetric unit with additionally 97 water molecules and two magnesium ions (

Acknowledgments

This article is dedicated to the memory of Carolina Sarah Förster. The work was supported within the RiNA network for RNA technologies by the Federal Ministry of Education and Research, the City of Berlin, and the European Regional Development Fund. We thank the Fonds der Chemischen Industrie (Verband der Chemischen Industrie e.V.) and the BMBF/VDI financed BiGRUDI network of the Robert Koch Institute (Berlin) for additional support. We gratefully acknowledge the Elettra synchrotron facility,

References (30)

  • J. Normanly et al.

    Eight base changes are sufficient to convert a leucine-inserting tRNA into a serine-inserting tRNA

    Proc. Natl. Acad. Sci. USA

    (1992)
  • P. Schimmel

    Aminoacyl tRNA synthetases: general scheme of structure-function relationships in the polypeptides and recognition of transfer RNAs

    Annu. Rev. Biochem.

    (1987)
  • D. Schatz et al.

    Interaction of Escherichia coli tRNASer with its cognate aminoacyl-tRNA synthetase as determined by footprinting with phosphorothioate-containing tRNA transcripts

    Proc. Natl. Acad. Sci. USA

    (1991)
  • S. Bilokapic et al.

    Structure of the unusual seryl-tRNA synthetase reveals a distinct zinc-dependent mode of substrate recognition

    EMBO J.

    (2006)
  • V. Biou et al.

    The 2.9 Å crystal structure of T. thermophilus seryl-tRNA synthetase complexed with tRNASer

    Science

    (1994)
  • Cited by (0)

    View full text