RESUMO
The position of the tertiary Levitt pair between nucleotides 15 and 48 in the transfer RNA core region suggests a key role in stabilizing the joining of the two helical domains, and in maintaining the relative orientations of the D and variable loops. E. coli tRNA(Gln) possesses the canonical Pu15-Py48 trans pairing at this position (G15-C48), while the tRNA(Cys) species from this organism instead features an unusual G15-G48 pair. To explore the structural context dependence of a G15-G48 Levitt pair, a number of tRNA(Gln) species containing G15-G48 were constructed and evaluated as substrates for glutaminyl and cysteinyl-tRNA synthetases. The glutaminylation efficiencies of these mutant tRNAs are reduced by two to tenfold compared with native tRNA(Gln), consistent with previous findings that the tertiary core of this tRNA plays a role in GlnRS recognition. Introduction of tRNA(Cys) identity nucleotides at the acceptor and anticodon ends of tRNA(Gln) produced a tRNA substrate which was efficiently aminoacylated by CysRS, even though the tertiary core region of this species contains the tRNA(Gln) G15-C48 pair. Surprisingly, introduction of G15-G48 into the non-cognate tRNA(Gln) tertiary core then significantly impairs CysRS recognition. By contrast, previous work has shown that CysRS aminoacylates tRNA(Cys) core regions containing G15-G48 with much better efficiency than those with G15-C48. Therefore, tertiary nucleotides surrounding the Levitt pair must significantly modulate the efficiency of aminoacylation by CysRS. To explore the detailed nature of the structural interdependence, crystal structures of two tRNA(Gln) mutants containing G15-G48 were determined bound to GlnRS. These structures show that the larger purine ring of G48 is accommodated by rotation into the syn position, with the N7 nitrogen serving as hydrogen bond acceptor from several groups of G15. The G15-G48 conformations differ significantly compared to that observed in the native tRNA(Cys) structure bound to EF-Tu, further implicating an important role for surrounding nucleotides in maintaining the integrity of the tertiary core and its consequent ability to present crucial recognition determinants to aminoacyl-tRNA synthetases.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Conformação de Ácido Nucleico , RNA de Transferência/química , RNA de Transferência/metabolismo , Aminoacil-tRNA Sintetases/química , Pareamento de Bases/genética , Sequência de Bases , Sítios de Ligação , Catálise , Cristalização , Cristalografia por Raios X , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Estabilidade de RNA , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA de Transferência/genética , RNA de Transferência de Cisteína/química , RNA de Transferência de Cisteína/genética , RNA de Transferência de Cisteína/metabolismo , RNA de Transferência de Glutamina/química , RNA de Transferência de Glutamina/genética , RNA de Transferência de Glutamina/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Guided by an in vitro selection experiment designed to obtain tight binding aptamers of Escherichia coli glutamine specific tRNA (tRNAGln) for glutaminyl-tRNA synthetase (GlnRS), we have engineered a tRNA mutant in which the five-nucleotide variable loop sequence 5'-44CAUUC48-3' is replaced by 5'-44AGGU48-3'. This mutant tRNA binds to GlnRS with 30-fold improved affinity compared to the wild type. The 2.7 A cocrystal structure of the RNA aptamer-GlnRS complex reveals major rearrangements in the central tertiary core of the tRNA, while maintaining an RNA-protein interface identical to the wild type. The repacked RNA core features a novel hydrogen bonding arrangement of the trans Levitt pair G15-U48, a new sulfate binding pocket in the major groove, and increased hydrophobic stacking interactions among the bases. These data suggest that enhanced protein binding to a mutant globular RNA can arise from stabilization of RNA tertiary interactions rather than optimization of RNA-protein contacts.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Conformação de Ácido Nucleico , RNA de Transferência de Glutamina/química , RNA de Transferência de Glutamina/metabolismo , Aminoacil-tRNA Sintetases/química , Pareamento de Bases/genética , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/enzimologia , Escherichia coli/genética , Biblioteca Gênica , Engenharia Genética , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Oligorribonucleotídeos/química , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica , RNA de Transferência de Glutamina/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Sulfatos/metabolismo , TermodinâmicaRESUMO
Preparation of large quantities of RNA molecules of a defined sequence is a prerequisite for biophysical analysis, and is particularly important to the determination of high-resolution structure by X-ray crystallography. We describe improved methods for the production of multimilligram quantities of homogeneous tRNAs, using a combination of chemical synthesis and enzymatic approaches. Transfer RNA half-molecules with a break in the anticodon loop were chemically synthesized on a preparative scale, ligated enzymatically, and cocrystallized with an aminoacyl-tRNA synthetase, yielding crystals diffracting to 2.4 A resolution. Multimilligram quantities of tRNAs with greatly reduced 3' heterogeneity were also produced via transcription by T7 RNA polymerase, utilizing chemically modified DNA half-molecule templates. This latter approach eliminates the need for large-scale plasmid preparations, and yields synthetase cocrystals diffracting to 2.3 A resolution at much lower RNA:protein stoichiometries than previously required. These two approaches developed for a tRNA-synthetase complex permit the detailed structural study of "atomic-group" mutants.