Gota Kawai

Structural requirement for the two-step dimerization of human immunodeficiency virus type 1 genome

Generation of RNA dimeric form of the human immunodeficiency virus type 1 (HIV-1) genome is crucial for viral replication. The dimerization initiation site (DIS) has been identified as a primary sequence that can form a stem-loop structure with a self-complementary sequence in the loop and a bulge in the stem. It has been reported that HIV-1 RNA fragments containing the DIS form two types of dimers, loose dimers and tight dimers. The loose dimers are spontaneously generated at the physiological temperature and converted into tight dimers by the addition of nucleocapsid protein NCp7. To know the biochemical process in this two-step dimerization reaction, we chemically synthesized a 39-mer RNA covering the entire DIS sequence and also a 23-mer RNA covering the self-complementary loop and its flanking stem within the DIS. Electrophoretic dimerization assays demonstrated that the 39-mer RNA reproduced the two-step dimerization process, whereas the 23-mer RNA immediately formed the tight dimer. Furthermore, deletion of the bulge from the 39-mer RNA prevented the NCp7-assisted tight-dimer formation. Therefore, the whole DIS sequence is necessary and sufficient for the two-step dimerization. Our data suggested that the bulge region regulates the stability of the stem and guides the DIS to the two-step dimerization process.

Polyamine Stimulation of the Synthesis of Oligopeptide-binding Protein (OppA) INVOLVEMENT OF A STRUCTURAL CHANGE OF THE SHINE-DALGARNO SEQUENCE AND THE INITIATION CODON AUG IN OppA mRNA

We previously suggested that the degree of polyamine stimulation of oligopeptide-binding protein (OppA) synthesis is dependent on the secondary structure and position of the Shine-Dalgarno (SD) sequence of OppA mRNA. To study the structural change of OppA mRNA induced by polyamines and polyamine stimulation of initiation complex formation, four different 130-mer OppA mRNAs containing the initiation region were synthesized in vitro.The structural change of these mRNAs induced by polyamines was examined by measuring their sensitivity to RNase T1, specific for single-stranded RNA, and RNase V1, which recognizes double-stranded or stacked RNA. In parallel, the effect of spermidine on mRNA-dependent fMet-tRNA binding to ribosomes was examined. Our results indicate that the secondary structure of the SD sequence and initiation codon AUG is important for the efficiency of initiation complex formation and that spermidine relaxes the structure of the SD sequence and the initiation codon AUG. The existence of a GC-rich double-stranded region close to the SD sequence is important for spermidine stimulation of fMet-tRNA binding to ribosomes. Spermidine apparently binds to this GC-rich stem and causes a structural change of the SD sequence and the initiation codon, facilitating an interaction with 30 S ribosomal subunits.

Crystal structure of Hfq from Bacillus subtilis in complex with SELEX-derived RNA aptamer: insight into RNA-binding properties of bacterial Hfq

Bacterial Hfq is a protein that plays an important role in the regulation of genes in cooperation with sRNAs. Escherichia coli Hfq (EcHfq) has two or more sites that bind RNA(s) including U-rich and/or the poly(A) tail of mRNA. However, functional and structural information about Bacillus subtilis Hfq (BsHfq) including the RNA sequences that specifically bind to it remain unknown. Here, we describe RNA aptamers including fragment (AG)3A that are recognized by BsHfq and crystal structures of the BsHfq–(AG)3A complex at 2.2 Å resolution. Mutational and structural studies revealed that the RNA fragment binds to the distal site, one of the two binding sites on Hfq, and identified amino acid residues that are critical for sequence-specific interactions between BsHfq and (AG)3A. In particular, R32 appears to interact with G bases in (AG)3A. Poly(A) also binds to the distal site of EcHfq, but the overall RNA structure and protein–RNA interaction patterns engaged in the R32 residues of BsHfq–(AG)3A differ from those of EcHfq–poly(A). These findings provide novel insight into how the Hfq homologue recognizes RNA.

The minimal tRNA: unique structure of Ascaris suum mitochondrial tRNASerUCU having a short T arm and lacking the entire D arm

The tertiary structure of Ascaris suum mitochondrial tRNASer UCU was examined by nuclear magnetic resonance analysis using its transcript, since tRNASer UCU, lacking the D arm and possessing a truncated T arm, is the shortest of all the known tRNAs. Most basepairs in the proposed secondary structure of tRNASer UCU were shown to exist, but the connector region comprising the truncated D loop and the extra loop was flexible. This flexibility, would enable adjustment of the mutual distance between the 3′‐terminus and the anticodon consistent with that of usual tRNAs. Thus, tRNASerUCU appears to function in a similar way to that of usual tRNAs in the ribosome.

Solution structure of a tmRNA-binding protein, SmpB, from Thermus thermophilus

Small protein B (SmpB) is required for trans‐translation, binding specifically to tmRNA. We show here the solution structure of SmpB from an extremely thermophilic bacterium, Thermus thermophilus HB8, determined by heteronuclear nuclear magnetic resonance methods. The core of the protein consists of an antiparallel β‐barrel twisted up from eight β‐strands, each end of which is capped with the second or third helix, and the first helix is located beside the barrel. Its C‐terminal sequence (20 residues), which is rich in basic residues, shows a poorly structured form, as often seen in isolated ribosomal proteins. The results are discussed in relation to the oligonucleotide binding fold.

Formation of a Complex Containing ATP, Mg2+, and Spermine STRUCTURAL EVIDENCE AND BIOLOGICAL SIGNIFICANCE

The conformation of ATP in the presence of Mg2+ and/or spermine was studied by 31P and 1H NMR, to clarify how polyamines interact with ATP. Spermine predominantly interacted with the β- and γ-phosphates of ATP in the presence of Mg2+. A conformational change of the β- and γ-phosphate of ATP with spermine could not be observed in the absence of Mg2+ by 31P NMR. It was found by1H NMR that the conformation of adenosine moiety of ATP was not influenced significantly by spermine. The binding of Mg2+ to ATP was slightly inhibited by spermine andvice versa. The results indicate that the binding sites of Mg2+ and spermine on ATP only partially overlap. The PotA protein, an ATP-dependent enzyme, was used as a model system to study the biological role of the ATP-Mg2+-spermine complex. The ATPase activity of PotA was greatly enhanced by spermine. Double reciprocal plots at several concentrations of spermine as an activator indicate that spermine interacts with ATP, but not with PotA. The activity of protein kinase A was also stimulated about 2-fold by spermine. The results suggest that a ternary complex of ATP-Mg2+-spermine may play an important role in some ATP-dependent reactions in vivo and in the physiological effects of endogenous polyamines.

Prediction of RNA Pseudoknots Using Heuristic Modeling with Mapping and Sequential Folding

Predicting RNA secondary structure is often the first step to determining the structure of RNA. Prediction approaches have historically avoided searching for pseudoknots because of the extreme combinatorial and time complexity of the problem. Yet neglecting pseudoknots limits the utility of such approaches. Here, an algorithm utilizing structure mapping and thermodynamics is introduced for RNA pseudoknot prediction that finds the minimum free energy and identifies information about the flexibility of the RNA. The heuristic approach takes advantage of the 5′ to 3′ folding direction of many biological RNA molecules and is consistent with the hierarchical folding hypothesis and the contact order model. Mapping methods are used to build and analyze the folded structure for pseudoknots and to add important 3D structural considerations. The program can predict some well known pseudoknot structures correctly. The results of this study suggest that many functional RNA sequences are optimized for proper folding. They also suggest directions we can proceed in the future to achieve even better results.

Conformational Rigidity of N4-Acetyl-2′-O-methylcytidine Found in tRNA of Extremely Thermophilic Archaebacteria (Archaea)

Abstract The conformational characteristics of N 4-acetyl-2′-O-methylcytidine (ac4Cm), which is one of the modified cytidines unique to the tRNA of extremely thermophilic archaebacteria, and related nucleosides, N 4-acetylcytidine (ac4C), 2′-O-methylcytidine (Cm) and cytidine, were analyzed by proton nuclear magnetic resonance spectroscopy. Ribose methylation and N 4-acylation were found to confer high conformational rigidity to the ribose moiety, suggesting that these post-transcriptional modifications play a role in structural stabilization of tRNA, which is of particular importance at high temperature.

Redox-coupled Conformational Alternations in Cytochrome c3 from D. vulgaris Miyazaki F on the Basis of its Reduced Solution Structure

Heteronuclear NMR spectroscopy was performed to determine the solution structure of (15)N-labeled ferrocytochrome c(3) from Desulfovibrio vulgaris Miyazaki F (DvMF). Although the folding of the reduced cytochrome c(3) in solution was similar to that of the oxidized one in the crystal structure, the region involving hemes 1 and 2 was different. The redox-coupled conformational change is consistent with the reported solution structure of D. vulgaris Hildenborough ferrocytochrome c(3), but is different from those of other cytochromes c(3). The former is homologous with DvMF cytochrome c(3) in amino acid sequence. Small displacements of hemes 1 and 2 relative to hemes 3 and 4 were observed. This observation is consistent with the unusual behavior of the 2(1)CH(3) signal of heme 3 reported previously. As shown by the (15)N relaxation parameters of the backbone, a region between hemes 1 and 2 has more flexibility than the other regions. The results of this work strongly suggest that the cooperative reduction of hemes 1 and 2 is based on the conformational changes of the C-13 propionate of heme 1 and the aromatic ring of Tyr43, and the interaction between His34 and His 35 through covalent and coordination bonds.

Conformatzonal Properties of a Novel Modified Nucleoside, 5-Formylcytidine, Found at the First Position of the Anticodon of Bovine Mitochondrial tRNAMet

Abstract Conformational properties of a novel modified nucleoside, 5-formylcytidine (f5C), which is found at the first position of the anticodon of bovine mitochondrial tRNAMet, were analyzed by 1H-NMR spectroscopy. f5C has a normal amino tautomeric form at position 4 of the base moiety. The results indicate the presence of an intramolecular hydrogen bond between the carbony1 of the 5-formyl group and the 4-amino function. f5C was found to exhibit the C3′-endo conformation exclusively and the enthalpy difference (ΔH) between the C2′-endo and C3′- endo forms was found to be 1.56 ± 0.13 kcal/mol, indicating f5C to be one of the most conformationally rigid nucleosides yet analyzed. The conformational rigidity of f5C may contribute to regulation of codon recognition by tRNAMet.