ABSTRACT
The RNA I modulator protein (Rom) acts as a co-regulator of ColE1 plasmid copy number by binding to RNA kissing hairpins and stabilizing their interaction. The structure of Rom has been determined in a new crystal form from X-ray diffraction data to 2.5 A resolution. In this structure, a dimer of the 57-amino-acid protein is found in the asymmetric unit. Each subunit consists almost entirely of two antiparallel alpha-helices joined by a short hairpin bend. The dimer contains a non-crystallographic twofold axis and forms a highly regular four-alpha-helical bundle. The structural packing in this novel crystal form is different from previously known Rom structures. The asymmetric unit contains one dimer, giving a crystal volume per protein weight (V(M)) of 1.83 A(3) Da(-1) and a low solvent content of 30%. Strong packing interactions and low solvation are characteristic of the structure. The Rom protein was cocrystallized with the Tar-Tar* kissing hairpin RNA. Although the electron-density maps do not show bound RNA, altered conformations in the side chains of Rom that are known to be involved in RNA binding have been identified. These results provide additional information about Rom protein conformational flexibility and suggest that the presence of a highly charged polymer such as RNA can promote tight packing of an RNA-binding protein, even when the RNA itself is not observed in the crystal.
Subject(s)
Bacterial Proteins/chemistry , Models, Molecular , RNA-Binding Proteins/chemistry , Crystallography, X-Ray , Dimerization , Escherichia coli Proteins/chemistry , RNA/chemistryABSTRACT
The crystal structure of the RNA octamer, 5'-GGCGUGCC-3' has been determined from x-ray diffraction data to 1.5 angstroms resolution. In the crystal, this oligonucleotide forms five self-complementary double-helices in the asymmetric unit. Tandem 5'GU/3'UG basepairs comprise an internal loop in the middle of each duplex. The NMR structure of this octameric RNA sequence is also known, allowing comparison of the variation among the five crystallographic duplexes and the solution structure. The G.U pairs in the five duplexes of the crystal form two direct hydrogen bonds and are stabilized by water molecules that bridge between the base of guanine (N2) and the sugar (O2') of uracil. This contrasts with the NMR structure in which only one direct hydrogen bond is observed for the G.U pairs. The reduced stability of the r(CGUG)2 motif relative to the r(GGUC)2 motif may be explained by the lack of stacking of the uracil bases between the Watson-Crick and G.U pairs as observed in the crystal structure.
Subject(s)
Base Pairing , Dinucleoside Phosphates/chemistry , Models, Chemical , Models, Molecular , RNA/chemistry , RNA/ultrastructure , Tandem Repeat Sequences , Computer Simulation , Crystallography , Magnetic Resonance Spectroscopy , Nucleic Acid ConformationABSTRACT
The initial aim of the Berkeley Structural Genomics Center is to obtain a near-complete structural complement of two minimal organisms, closely related pathogens Mycoplasma genitalium and M. pneumoniae. The former has fewer than 500 genes and the latter fewer than 700 genes. To achieve this goal, the current protein targets have been selected starting with those predicted to be most tractable and likely to yield new structural and functional information. During the past 3 years, the semi-automated structural genomics pipeline has been set up from cloning, expression, purification, and ultimately to structural determination. The results from the pipeline substantially increased the coverage of the protein fold space of M. pneumoniae and M. genitalium. Furthermore, about 1/2 of the structures of 'unique' protein sequences revealed new and novel folds, and over 2/3 of the structures of previously annotated 'hypothetical proteins' inferred their molecular functions.
Subject(s)
Bacterial Proteins/genetics , Genome, Bacterial/genetics , Models, Molecular , Mycoplasma genitalium/genetics , Mycoplasma pneumoniae/genetics , Protein Folding , Proteomics/methods , Cloning, Molecular , CrystallizationABSTRACT
We have determined the crystal structure, at 1.4A, of the Nudix hydrolase DR1025 from the extremely radiation resistant bacterium Deinococcus radiodurans. The protein forms an intertwined homodimer by exchanging N-terminal segments between chains. We have identified additional conserved elements of the Nudix fold, including the metal-binding motif, a kinked beta-strand characterized by a proline two positions upstream of the Nudix consensus sequence, and participation of the N-terminal extension in the formation of the substrate-binding pocket. Crystal structures were also solved of DR1025 crystallized in the presence of magnesium and either a GTP analog or Ap(4)A (both at 1.6A resolution). In the Ap(4)A co-crystal, the electron density indicated that the product of asymmetric hydrolysis, ATP, was bound to the enzyme. The GTP analog bound structure showed that GTP was bound almost identically as ATP. Neither nucleoside triphosphate was further cleaved.
