Three-dimensional structure of a mutant ribonuclease T1 (Y45W) complexed with non-cognizable ribonucleotide, 2'AMP, and its comparison with a specific complex with 2'GMP.

The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a non-cognizable ribonucleotide, 2'AMP, has been determined and refined to an R-factor of 0.159 using X-ray diffraction data at 1.7 A resolution. A specific complex of the enzyme with 2'GMP was also determined and refined to an R-factor of 0.173 at 1.9 A resolution. The adenine base of 2'AMP was found at a base-binding site that is far apart from the guanine recognition site, where the guanine base of 2'GMP binds. The binding of the adenine base is mediated by a single hydrogen bond and stacking interaction of the base with the imidazole ring of His92. The mode of stacking of the adenine base with His92 is similar to the stacking of the guanine base observed in complexes of ribonuclease T1 with guanylyl-2',5'-guanosine, reported by Koepke et al., and two guanosine bases, reported by Lenz et al., and in the complex of barnase with d(GpC), reported by Baudet & Janin. These observations suggest that the site is non-specific for base binding. The phosphate group of 2'AMP is tightly locked at the catalytic site with seven hydrogen bonds to the enzyme in a similar manner to that of 2'GMP. In addition, two hydrogen bonds are formed between the sugar moiety of 2'AMP and the enzyme. The 2'AMP molecule adopts the anti conformation of the glycosidic bond and C-3'-exo sugar pucker, whereas 2'GMP is in the syn conformation with C-3'-endo-C'-2'-exo pucker. The mutation enhances the binding of 2'GMP with conformational changes of the sugar ring and displacement of the phosphate group towards the interior of the catalytic site from the corresponding position in the wild-type enzyme complex. Comparison of two crystal structures obtained provides a solution to the problem that non-cognizable nucleotides exhibit unexpectedly strong binding to the enzyme, compared with high specificity in nucleolytic activity. The results indicate that the discrimination of the guanine base from the other nucleotide bases at the guanine recognition site is more effective than that estimated from nucleotide-binding experiments so far.     

Product analyses in DNA strand scission by antitumor antibiotic elsamicin A.

Elsamicin A is an antitumor antibiotic with fascinating chemical structure and a good candidate for pharmaceutical development. Molecular mechanism of DNA backbone cleavage mediated by Fe(II)-elsamicin A has been examined. Product analysis using DNA sequencing gels and HPLC reveals the production of damaged DNA fragments bearing 3'-/5'-phosphate and 3'-phosphoglycolate termini associated with formation of free base. In addition, hydrazine-trapping experiments indicate that C-4' hydroxylated abasic sites are formed concomitant with DNA degradation by Fe(II)-elsamicin A. The results lead to the conclusion that the hydroxyl radical formed in Fe(II)-elsamicin A plus dithiothreitol system oxidizes the deoxyribose moiety via hydrogen abstraction predominantly at the C-4' carbon of the deoxyribose backbone and ultimately produces strand breakage of DNA.     

Addition of an extra substrate binding site and partial destabilization of stem structures in HDV ribozyme give rise to high sequence-specificity for its target RNA

Because the substrate binding site (P1) of HDV ribozyme consists of only seven nucleotides, cleavage of undesired RNA is likely to occur when applied for a specific long RNA target such as mRNA. To overcome this problem, we designed modified trans-acting HDV ribozymes with an extra substrate-binding site (P5) in addition to the original binding site (P1). By inserting an additional seven base-pair stem (P5 stem) into the J1/2 single-stranded region of the ribozyme core system and partial destabilization of the P2 or P4 stem, we succeeded in preparation of new HDV ribozymes that can cleave the target RNA depending on the formation of P5 stem. Moreover, the ribozyme with a six-nucleotide P1 site was able to distinguish the substrate RNA with a complete match from that with a single mismatch in the P1 region. These results suggest that the HDV ribozyme system is useful for the application in vivo.     

Constitutive MAP kinase activation in hematopoietic stem cells induces a myeloproliferative disorder

Myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPNs) are a group of myeloid neoplasms in which abnormal activation of the Ras signaling pathway is commonly observed. The PI3K/Akt pathway is a known target of Ras; however, activation of the PI3K/Akt pathway has been shown to lead to neoplastic transformation of not only myeloid but also lymphoid cells, suggesting that pathways other than the PI3K/Akt pathway should play a central role in pathogenesis of Ras-mediated MDS/MPN. The MEK/ERK pathway is another downstream target of Ras, which is involved in regulation of cell survival and proliferation. However, the role of the MEK/ERK pathway in the pathogenesis of MDS/MPN remains unclear. Here, we show that introduction of a constitutively activated form of MEK into hematopoietic stem cells (HSCs) causes hematopoietic neoplasms that are limited to MDS/MPNs, despite the multipotent differentiation potential of HSCs. Active MEK-mediated MDS/MPNs are lethal, but are not considered a frank leukemia because it cannot be transplanted into naïve animals. However, transplantation of MDS/MPNs co-expressing active MEK and an anti-apoptotic molecule, Bcl-2, results in T-cell acute lymphocytic leukemia (T-ALL), suggesting that longevity of cells may impact transplantability and alter disease phenotype. Our results clearly demonstrate the proto-oncogenic property of the MEK/ERK pathway in hematopoietic cells, which manifest in MDS/MPN development.     

27 amino acid residues can be deleted from the N-terminus of human lymphotoxin without impairment of its cytotoxic activity

In order to study the relationship between activity and structure of human lymphotoxin (hLT, 171 aa), we synthesized the gene (519 bp) for hLT and expressed it in Escherichia coli. Purification of the recombinant hLT from crude extracts was difficult because of the low level of expression of the gene. To improve the yield of the recombinant protein, we prepared five truncated genes for mutant proteins in which 25, 26, 27, 28 and 37 amino acid residues, respectively, were missing from the N-terminus. All of the genes were efficiently expressed and adequate amounts of mutant proteins were synthesized. The proteins were recovered mainly in the supernatant fractions after disruption of cells, with the exception of LTδ37N, in which 37 residues were absent from the N-terminal region. Cytotoxic activities against mouse fibroblast L929 cells were detected in supernatant fractions that contained these mutant proteins, except in the case of LTδ28N, which lacks the first amino acid residue conserved in both hLT and human tumour necrosis factor (hTNF). LTδ27N, which is the smallest of the active proteins, was purified to homogeneity, and its cytotoxic activity was found to be similar to that of recombinant hTNF.     

NMR Studies of a Lead Ribozyme and Its Non-Cleavable Analogue

Abstract

The structure of a lead ribozyme, which consists of two RNA strands, at neutral pH has been studied by NMR. Nearly all resonances of imino protons, base protons (H2, H5, H6 and H8) and sugar protons (H1′ and H2′) were assigned sequentially. Interesting structural features which deviate from the standard structure were found for the residues at an active site which consists of an internal loop. No indication of stable G:A base pairs was found in the loop. The effect of addition of Pb²⁺ was studied by the use of a non-cleavable analogue in which the cytidine at a cleavage site is replaced by 2′-O-methylcytidine. It was suggested that Pb²⁺ binds close to the cleavage site and that the structural change induced by Pb²⁺ is moderate and localized.

     

Fabrication of doughnut-shaped particles from spheroidal paramylon granules of Euglena gracilis using acetylation reaction

An anhydrous type of paramylon, the micro-sized granular storage carbohydrate (β-1,3-glucan) of Euglena, was transformed from a spheroidal to a doughnut-like shape by acetylation. Fourier transform infrared spectroscopic measurements suggested that the doughnut formation is due to removal of accessible regions of paramylon particles by acetylation of glucans. A time-course observation of the paramylon granules during acetylation by using field emission scanning electron microscopy revealed that the doughnut-making process begins with the removal of an outer membrane of the granule and that the central region of the granules is preferentially removed with the survival of a thick rim part to give the doughnut-like particles.     

Structural Characterization of a DNA Duplex Modeled on a DNA:RNA Hybrid of the Polypurine Tract Recognized by a Reverse Transcriptase

Abstract

The structure of d(TTAAAAGAAAAGGG):d(CCCTTTTCTTTTAA) has been characterized by NMR. The minor grooves of the two dA-tracts are suggested to be rather narrow, and the portion linking the two dA tracts exhibits a slightly deviated structure from a standard B DNA, in order to maintain the narrowness of the minor groove. The structure of the dG-tract is also slightly deviated. Additionally, specific broadening of resonances is observed for the residues at or near the junction between the dA-tract and the dG-tract, suggesting local structural polymorphology.

     

Interaction of (2′ -5′) and (3′ -5′) Linked 2-Aminoadenylyl-3-aminoadenosines with Polyuridylic Acid

Abstract

2′-5′ and 3′-5′ linked 2-aminoadenylyl-2-aminoadenosines [(2′-5′)n²Apn²A (1) and (3′-5′)n²Apn²A (2)] were synthesized by condensation of 5′-O-monomethoxytrityl-N ² N ⁶-dibenzoyl-2-aminoadenosine and N ²,N ⁶,2′,3′-O-tetrabenzoyl-2-aminoadenosine 5′-phosphate using dicyclohexylcarbodiimide (DCC). The conformational properties of these dimers 1 and 2 were examined by UV, NMR and CD spectroscopy. The results reveal that the 2′-5′-isomer 1 takes a stacked conformation, which contains a larger base-base overlap and is more stable against thermal perturbation with respect to the 3′-5′-isomer 2. Interactions of 1 and 2 with polyuridylic acid (Poly (U)) were also examined by Tm, mixing curves, UV and CD spectra. Both the dinucleoside isomers 1 and 2 formed a complex of 1 : 2 stoichiometry with poly(U), which was much more stable than that of the corresponding ApA isomer