ABSTRACT
The tRNA(Gly) and glycyl-tRNA synthetase (GlyRS) system is an evolutionary special case within the class II aminoacyl-tRNA synthetases because two divergent types of GlyRS exist: an archaebacterial/human type and an eubacterial type. The tRNA identity elements which determine the correct aminoacylation process are located in the aminoacyl domain of tRNA(Gly). To obtain further insight concerning structural investigation of the identity elements, the Escherichia coli seven-base-pair tRNA(Gly) acceptor-stem helix was crystallized. Data were collected to 2.0 A resolution using synchrotron radiation. Crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 35.35, c = 130.82 A, alpha = beta = 90, gamma = 120 degrees and two molecules in the asymmetric unit.
Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/isolation & purification , RNA, Transfer, Gly/analysis , RNA, Transfer, Gly/chemistry , X-Ray Diffraction/methods , Crystallization , Protein ConformationABSTRACT
Chemically synthesized RNAs with the unnatural L-configuration possess enhanced in vivo stability and nuclease resistance, which is a highly desirable property for pharmacological applications. For a structural comparison, both L- and D-RNA oligonucleotides of a shortened Thermus flavus 5S rRNA A-helix were chemically synthesized. The enantiomeric RNA duplexes were stochiometrically cocrystallized as a racemate, which enabled analysis of the D- and L-RNA enantiomers in the same crystals. In addition to a biochemical investigation, diffraction data were collected to 3.0 A resolution using synchrotron radiation. The crystals belonged to space group P3(1)21, with unit-cell parameters a = b = 35.59, c = 135.30 A, gamma = 120 degrees and two molecules per asymmetric unit.
Subject(s)
RNA, Ribosomal, 5S/analysis , RNA, Ribosomal, 5S/chemistry , X-Ray Diffraction/methods , Crystallization , Protein Structure, Secondary/physiology , StereoisomerismABSTRACT
In order to understand elongator tRNA(Ser) and suppressor tRNA(Sec) identity elements, the respective acceptor-stem helices have been synthesized and crystallized in order to analyse and compare their structures in detail at high resolution. The synthesis, crystallization and preliminary X-ray diffraction results for a seven-base-pair tRNA(Ser) acceptor-stem helix are presented here. Diffraction data were collected to 1.8 A, applying synchrotron radiation and cryogenic cooling. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 36.14, b = 38.96, c = 30.81 A, beta = 110.69 degrees .
Subject(s)
Oligonucleotides/chemistry , RNA, Transfer, Ser/chemistry , Base Sequence , Crystallization , Nucleic Acid Conformation , Oligonucleotides/chemical synthesis , X-Ray DiffractionABSTRACT
The pharmacokinetic properties of an aptamer against the tumour-marker protein tenascin-C have recently been successfully improved by the introduction of locked nucleic acids (LNAs) into the terminal stem of the aptamer. Since it is believed that this post-SELEX optimization is likely to provide a more general route to enhance the in vitro and in vivo stability of aptamers, elucidation of the structural basis of this improvement was embarked upon. Here, the crystallographic and X-ray diffraction data of the isolated aptamer stem encompassed in a six-base-pair duplex both with and without the LNA modification are presented. The obtained all-LNA crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 52.80, c = 62.83 angstroms; the all-RNA crystals belong to space group R32, with unit-cell parameters a = b = 45.21, c = 186.97 angstroms, gamma = 120.00 degrees.
Subject(s)
Nucleic Acids/chemistry , Peptide Fragments/chemistry , RNA/chemistry , Tenascin/chemistry , Binding Sites , Crystallization , Peptide Fragments/isolation & purification , Tenascin/isolation & purification , X-Ray DiffractionABSTRACT
The crystal structure of a serine-specific tRNA acceptor-stem microhelix, the binding site for the seryl-tRNA synthetase, was solved by X-ray analysis. This seven-base-pair tRNA(Ser) microhelix forms endless rows of helices in the crystal lattice, with two helices stacking 'head-to-head' onto each other, resulting in an intermolecular guanosine stacking of the first purine nucleotides at the 5'-strands of the tRNA(Ser) microhelices. A network of 75 water loci could be associated with each RNA duplex. Unusual local geometric backbone parameters could be detected in the region of the G4 phosphate located in the 5'-strand of the helix, which lead to a ;kink' in this region and to an irregularly bent helix. The role of the specific hydration pattern and of the irregular conformation of the tRNA(Ser) acceptor-stem helix is discussed and summarized.