Hepatitis 8 virus (HDV) contains a singlestranded circular RNA genome of 1.7 kilobases. In this report we demonstrate that subfragments of HDV RNA can undergo autocatalytic cleavage. This cleavage requires at least 500 pM of Mg2+ or Ca2+, is not affected by varying the pH from 5.0 to 9.1, and occurs with RNA fragments as small as 133 nucleotides. The larger RNA fragments containing additional HDV sequences have a lower efficiency of cleavage. Deletion analysis at both ends of RNA subfragments suggested that the catalytic ability of HDV RNA resides in a stretch of no more than 117 nucleotides around the cleavage site. The cleavage occurs at the phosphodiester bond between nucleotides 688 and 689 on the HDV genomic map, generating a 5' fragment with a terminal uridyl 2',3'-cyclic monophosphate residue and a 3' fragment with a guanosyl residue with a 5'-hydroxyl group. The smallest autocleaving RNA does not contain the "hammerhead" sequence required for the autocleavage of other known selfcleaving RNA. The cleavage of HDV RNA occurs at a much faster rate, even at a very low Mg2+ concentration, than that of other "ribozymes." Thus, HDV RNA represents a distinct class of ribozyme.Human hepatitis 8 virus (HDV) is a defective virus often associated with fulminant hepatitis in hepatitis B virus carriers. The virus particles have an envelope in which hepatitis B virus surface antigen is located. Internal to the envelope are HDV-specific 6 antigen and a single-stranded circular RNA genome of 1.7 kilobases (kb) (1-3). This animal virus is distinctive because it has such a mixed structure that resembles somewhat the virus coat protein-encapsidated plant virus satellite and virusoid RNAs. The HDV RNA possesses several additional properties similar to those of circular RNA of viroids; for instance, the presence of a high degree of intramolecular self-complementarity and conservation of the consensus sequences known to be important for viroid RNA replication (4,5). However, HDV RNA is much larger than viroid RNAs and encodes at least one structural protein (6 antigen), whereas viroid RNA does not encode any protein. Despite these differences HDV RNA and viroid RNA appear to replicate through a similar mechanism-i.e., a rolling circle mechanism (6)-since a larger-than-genomic size RNA intermediate has been detected in HDV-infected cells (7,8). Thus a mechanism must exist to cleave these RNA intermediates into monomeric RNA and to circularize them.One viroid and several virusoid and satellite RNAs have been shown to possess an autocleaving activity (9-11). The cleavage sites of these RNAs correspond to sites of in vivo RNA processing, suggesting that these autocleavage reactions have physiological roles. A linear dimer cDNA of HDV genome can be processed into a monomeric RNA when transfected into a monkey kidney (COS) cell line (unpublished observation), suggesting either that HDV RNA has an unusual secondary structure that allows for specific attack by a cellular RNase or that HDV RNA has a specific autocleavage activit...

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The functional and structural significance of the C-terminal region of Thermoanaerobacter ethanolicus 39E amylopullulanase (TetApu) was explored using C-terminal truncation mutagenesis. Comparative studies between the engineered full-length (TetApuM955) and its truncated mutant (TetApuR855) included initial rate kinetics, fluorescence and CD spectrometric properties, substrate-binding and hydrolysis abilities, thermostability, and thermodenaturation kinetics. Kinetic analyses revealed that the overall catalytic efficiency, k (cat)/K (m), was slightly decreased for the truncated enzymes toward the soluble starch or pullulan substrate. Changes to the substrate affinity, K (m), and turnover rate, k (cat), varied in different directions for both types of substrates between TetApuM955 and TetApuR855. TetApuR855 exhibited a higher thermostability than TetApuM955, and retained similar substrate-binding ability and hydrolyzing efficiency against the raw starch substrate as TetApuM955 did. Fluorescence spectroscopy indicated that TetApuR855 retained an active folding conformation similar to TetApuM955. A CD-melting unfolding study was able to distinguish between TetApuM955 and TetApuR855 by the higher apparent transition temperature in TetApuR855. These results indicate that up to 100 amino acid residues, including most of the C-terminal fibronectin typeIII (FnIII) motif of TetApuM955, could be further removed without causing a seriously aberrant change in structure and a dramatic decrease in soluble starch and pullulan hydrolysis.

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The three-dimensional solution structure of the ligand binding D2 domain of the fibroblast growth factor receptor (FGFR) is determined using multidimensional NMR techniques. The atomic root-mean-square distribution for the backbone atoms in the structured region is 0.64 A. Secondary structural elements in the D2 domain include 11 beta-strands arranged antiparallely into two layers of beta-sheets. The structure of the D2 domain is characterized by the presence of a short flexible helix that protrudes out of the layers of beta-sheets. Results of size exclusion chromatography and sedimentation velocity experiments show that the D2 domain exists in a monomeric state both in the presence and in the absence of bound sucrose octasulfate (SOS), a structural analogue of heparin. Comparison of the solution structure of the D2 domain with the crystal structure of the protein (D2 domain) in the FGF signaling complex reveals significant differences, suggesting that ligand (FGF) binding may induce significant conformational changes in the receptor. SOS binding sites in the D2 domain have been mapped on the basis of the 1H-15N chemical shift perturbation data. SOS binds to the positively charged residues located in beta-strand III and the flexible helix. Isothermal titration calorimetry data indicate that the ligand (hFGF-1) binds strongly (Kd approximately 10(-9) M) to the D2 domain even in the absence of SOS. Binding of SOS to either the D2 domain or hFGF-1 does not seem to be the driving force for the formation of the D2-hFGF-1 binary complex. The function of SOS binding appears to stabilize the preformed D2-FGF binary complex.