NMR structure and preliminary crystallographic studies of small protein B (SmpB) from Aquifex aeolicus

textThe tmRNA-SmpB system is a highly conserved quality control system in all prokaryotes. It has the dual function of rescuing ribosomes stalled on defective mRNA templates and tagging proteins resulting from problematic messages for degradation. Small protein B (SmpB) is an essential component of this system, lacking significant homology with any known proteins other than the same proteins from different species. The structure of the SmpB from Aquifex aeolicus was determined by multidimensional NMR techniques. It consists of an antiparallel b-barrel, with three helices packed outside the core of the barrel. While the overall structure of SmpB appears to be unique, the protein does contain an embedded oligonucleotide binding (OB) fold; in this respect SmpB has similarity to several other RNA-binding proteins that are known to be associated with translation, including initiation factor 1 (IF1), ribosomal protein S17 and the N-terminal domain of aspartyltRNA synthetase (DRS). Conserved amino acids on the protein surface that are likely candidates for direct interactions with the tmRNA and other components of the translational apparatus were identified. The presence of the two widely separated clusters of conserved surface amino acids suggests that SmpB could function either by stabilizing two distal regions of the tmRNA, or by facilitating an interaction between the tmRNA and another component of the translational apparatus. While the C-terminal ~20 amino acids appear to be unstructured, their presence may be essential for the function of SmpB in the trans-translation process. The structural model reported in this dissertation will be essential in ultimately determining the detailed mechanism by which the tmRNASmpB system performs its functions. Results of preliminary NMR perturbation studies on the complexes between SmpB and several RNA molecules are described. In addition, single crystals of the core fragment of the SmpB were obtained by the vapor diffusion method in sitting-drops at room temperature. Preliminary crystallographic analysis reveals that the crystal belongs to a tetragonal lattice, with unit cell parameters a = b = 55.0 Å, c = 65.9 Å, a = b = g = 90°. Further structural determinations by molecular rep.Biological Sciences, School o