Crystallization and preliminary X-ray diffraction studies of HutP protein: an RNA-binding protein that regulates the transcription of hut operon in Bacillus subtilis

HutP is an RNA-binding protein and regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis by binding to cis-acting regulatory sequences on hut mRNA. HutP and its mutant, which has increased affinity for the regulatory sequences, were purified and crystallized by the hanging-drop vapor diffusion method. The space group was P2(1)3 with unit cell dimensions a=b=c=95.6A for HutP and a=b=c=96.8A for the mutant. Complete data sets of 3.0-A resolution for wild-type HutP and of 2.70-A resolution for the mutant HutP were collected.     

A chloroplastic RNA-binding protein is a new member of the PPR family

P67, a new protein binding to a specific RNA probe, was purified from radish seedlings [Echeverria, M. and Lahmy, S. (1995) Nucleic Acids Res. 23, 4963–4970]. Amino acid sequence information obtained from P67 microsequencing allowed the isolation of genes encoding P67 in radish and Arabidopsis thaliana. Immunolocalisation experiments in transfected protoplasts demonstrated that this protein is addressed to the chloroplast. The RNA-binding activity of recombinant P67 was found to be similar to that of the native protein. A significant similarity with the maize protein CRP1 [Fisk, D.G., Walker, M.B. and Barkan, A. (1999) EMBO J. 18, 2621–2630] suggests that P67 belongs to the PPR family and could be involved in chloroplast RNA processing.     

Crystal structure-based exploration of the important role of Arg106 in the RNA-binding domain of human coronavirus OC43 nucleocapsid protein

Human coronavirus OC43 (HCoV-OC43) is a causative agent of the common cold. The nucleocapsid (N) protein, which is a major structural protein of CoVs, binds to the viral RNA genome to form the virion core and results in the formation of the ribonucleoprotein (RNP) complex. We have solved the crystal structure of the N-terminal domain of HCoV-OC43 N protein (N-NTD) (residues 58 to 195) to a resolution of 2.0 Å. The HCoV-OC43 N-NTD is a single domain protein composed of a five-stranded β-sheet core and a long extended loop, similar to that observed in the structures of N-NTDs from other coronaviruses. The positively charged loop of the HCoV-OC43 N-NTD contains a structurally well-conserved positively charged residue, R106. To assess the role of R106 in RNA binding, we undertook a series of site-directed mutagenesis experiments and docking simulations to characterize the interaction between R106 and RNA. The results show that R106 plays an important role in the interaction between the N protein and RNA. In addition, we showed that, in cells transfected with plasmids that encoded the mutant (R106A) N protein and infected with virus, the level of the matrix protein gene was decreased by 7-fold compared to cells that were transfected with the wild-type N protein. This finding suggests that R106, by enhancing binding of the N protein to viral RNA plays a critical role in the viral replication. The results also indicate that the strength of N protein/RNA interactions is critical for HCoV-OC43 replication.     

Crystallization and preliminary X-Ray analysis of recombinant staphylokinase

Diffraction quality crystals of recombinant staphylokinase (STAR) have been grown by the hanging drop vapor diffusion technique from a solution containing MgCl₂, Tris buffer (pH 8.5), and polyethylene glycol 4000. The crystals belong to the monoclinic space group C2 with unit cell dimensions a = 60.6 Å, b = 43.7 Å, c = 54.3 Å, and β = 115.6°. Å complete native data set to 1.8 Å resolution has been collected using synchrotron radiation. Proteins 27:160–161 © 1997 Wiley-Liss, Inc.     

Cellular levels of trp RNA-binding attenuation protein in Bacillus subtilis

Expression of the Bacillus subtilis trp genes is negatively regulated by an 11-subunit trp RNA-binding attenuation protein (TRAP), which is activated to bind RNA by binding l-tryptophan. We used Western blotting to estimate that there are 200 to 400 TRAP 11-mer molecules per cell in cells grown in either minimal or rich medium.     

Crystallization of the IIA domain of the glucose permease of Bacillus subtilis

The IIA domain of the glucose permease of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) from Bacillus subtilis has been crystallized. Crystals are obtained from ammonium sulfate solution. They diffract to at least 2.2 A resolution, and belong to space group C222(1), with unit cell dimensions: a = 74.2 A; b = 54.9 A; c = 67.0 A.     

Crystallization and preliminary X-ray crystallographic studies on Type III antifreeze protein.

Type III antifreeze protein, more specifically the recombinant QAE-Sephadex-binding isoform, has been crystallized in 50-55% saturated ammonium sulfate, 0.1 M sodium acetate, pH 4.0-4.5. The resultant crystals belong to the orthorhombic space group P212121 with a = 32.60 A, b = 39.00 A, and c = 46.57 A and diffract to at least 1.7 A. A set of 1.7-A native data has been collected, with completeness 93.4% and Rsym of 0.069. Initial screening for heavy-atom derivatives has yielded a Pt-bound derivative.     

Crystallization and preliminary X-ray diffraction studies of the cartilage link protein from bovine trachea

Cartilage extracellular matrix link protein, having molecular mass of approximately 40 kDa, is a metalloprotein that binds divalent cations and is only soluble in low ionic strength solutions. The link protein was purified from bovine trachea and has been crystallized by a vapor diffusion method using PEG 3350 as precipitant. The crystal symmetry is P1, and the unit cell dimensions are a = 43.55, b = 53.11, c = 60.10 Å, α = 90.44, β = 106.21, γ = 101.51°. The V_M of 1.8 Å₃/Da is consistent with the presence of two molecules of the link protein in the asymmetric unit. The crystals diffract X-rays from a synchrotron source to 1.7 Å resolution. © 1995 Wiley-Liss, Inc.     

cDNA encoding a wheat (Triticum aestivum cv. Chinese Spring) glycine-rich RNA-binding protein

A wheat cDNA encoding a glycine-rich RNA-binding protein, whGRP-1, was isolated. WhGRP-1 contains two conserved domains, the RNA-binding motif (RNP motif) combined with a series of glycine-rich imperfect repeats, characteristic of a conserved family of plant RNA-binding proteins. Northern analysis revealed that whGRP-1 mRNA accumulates to high levels in roots and to lower levels in leaves of wheat seedlings. whGRP-1 mRNA accumulation is not enhanced by exogenous abscisic acid in seedlings and accumulates to very high levels during wheat embryo development, showing a pattern different from that of the ABA-inducible wheat Em gene.     

HydRA: Deep-learning models for predicting RNA-binding capacity from protein interaction association context and protein sequence

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cDNA cloning and characterization of Drb1, a new member of RRM-type neural RNA-binding protein

Neural RNA recognition motif (RRM)-type RNA-binding proteins play essential roles in neural development. To search for a new member of neural RRM-type RNA-binding protein, we screened rat cerebral expression library with polyclonal antibody against consensus RRM sequences. We have cloned and characterized a rat cDNA that belongs to RRM-type RNA-binding protein family, which we designate as drb1. Orthologs of drb1 exist in human and mouse. The predicted amino acid sequence reveals an open reading frame of 476 residues with a corresponding molecular mass of 53kDa and consists of four RNA-binding domains. drb1 gene is specifically expressed in fetal (E12, E16) rat brain and gradually reduced during development. In situ hybridization demonstrated neuron-specific signals in fetal rat brain. RNA-binding assay indicated that human Drb1 protein possesses binding preference on poly(C)RNA. These results indicate that Drb1 is a new member of neural RNA-binding proteins, which expresses under spatiotemporal control.     

Crystallization of NAD+ synthetase from Bacillus subtilis

NAD⁺-synthetase is a ubiquitous enzyme catalyzing the last step in the biosynthesis of NAD⁺. Mutants of NAD⁺ synthetase with impaired cellular functions have been isolated, indicating a key role for this enzyme in cellular metabolism. Crystals of the enzyme from Bacillus subtilis suitable for x-ray crystallographic investigation have been grown from polyethylene glycol solutions. Investigation on the structural organization of NAD⁺ synthetase, an enzyme fundamental for NAD⁺ biosynthesis, and belonging to the recently characterized amidotransferase enzymatic family, will provide more insight into the catalytic mechanism of deamido-NAD⁺ → NAD⁺ conversion, a biosynthetic process that is a potential target for the development of antibiotic compounds against Bacillus sp. and related bacteria. © 1996 Wiley-Liss, Inc.