Nucleic acids symposium series (2004)最新文献

Transfer RNAs (tRNAs) are decorated with various post-transcriptional modifications which are enzymatically introduced at various stages of maturation. It is known that eukaryotic tRNAs are modified both in nucleus and cytoplasm. However, the order of tRNA modifications remains to be investigated. To unveil the precise timing of each modification associated with tRNA processing, we isolated precursor forms of Saccharomyces cerevisiae tRNAs at different stages of maturation, and analyzed their primary structures including modifications by mass spectrometry. The primary transcript of tRNA(Ile) was isolated from the tRNA fraction co-precipitated with Lhp1p, a yeast homolog of La protein. The precursor tRNA(Ile) without 3'-trailer sequence was isolated from the expression controllable strain when RNase P was transiently inactivated. Mass spectrometric analyses of these tRNAs revealed that many modifications were fully introduced at the stage of primary transcript, however, some modifications were partially introduced.

RsgA is a unique GTP hydrolytic protein, in which the GTPase activity is significantly enhanced by the small ribosomal subunit. Depletion of RsgA causes slow cell growth as well as defects in the subunit assembly of the ribosome and the 16S rRNA processing, suggesting its involvement in the maturation of the small subunit. Several antibiotics bound to the decoding center of the small subunit inhibited the ribosome-dependent GTPase activity of RsgA. Our recent study using chemical modification indicates that the binding of RsgA induces conformational changes around the A site, P site, and helix 44. These results suggest that RsgA is involved in the maturation step of the decoding center of the small subunit of ribosome. Here, we also show a physiological role of RsgA under stress condition.

Synthesis of benzoquinone ansa-adenosines, which are rationally designed as Hsp90 inhibitors by extracting and fusing a natural substrate, ATP, and a natural product, geldanamycin, was described. This simpler scaffold design provides practical synthesis of a set of analogs and demonstrates synthetic innovation.

We designed and synthesized a nucleoside derivative in which the nucleobase is replaced with acridone. The nucleoside derivative was incorporated into an oligodeoxyribonucleotide (ODN), and its influence on the stability of ODN hybrids and its fluorescent properties in a DNA duplex were measured by thermodynamic analysis and fluorescent spectroscopy. These results showed that the acridonyl group could distinguish the type of nucleobase paired from fluorescent intensity, although the hybrid stability did not depend significantly on the types of nucleobases paired.

Here we highlight the unusual substrate specificity of Plasmodium falciparum thymidylate kinase (PfTMK) and the validity of the enzyme as a new drug target. Furthermore, we predict that the Anaplasma marginale enzyme has attractive domain constituents and may be functionally different from other TMPKs. We postulate that thymidylate kinases could have multiple attractive functions in pathogens and may be a new drug target against numerous microorganisms.

In the early stages of the evolution of life, RNA-polypeptide complexes (RNPs) have been suggested to play crucial roles. At a certain developmental stage of ancient RNPs, their RNA and polypeptide components could evolve in an interdependent manner to develop complex structures and functions. To mimic this possible process, we have designed an RNA molecule that can act as a template for chemical peptide ligation. This designed RNA possesses two peptide-binding sites that capture the two basic peptides. The designed RNA actually facilitated the peptide ligation. The resulting ligated peptide, which has two RNA binding sites, can in turn function as a trans-activator that enhances the intrinsic ribozymatic activity of the designed RNA.

In Plasmodium falciparum, deoxyguanylate was found to be a substrate for several DNA metabolizing enzymes. Guanylate kinase utilizes dGMP with very low specificity, which is estimated to be the lowest among well-known prokaryotic and eukaryotic enzymes. Furthermore, thymidylate kinase, which is a pyrimidine specific enzyme, was found to phosphorylate dGMP with a surprisingly high specificity similar to that of the natural substrate. The above mentioned distinctions are specific for the Plasmodium protozoa and provide an interesting method for tracking dGMP metabolism during development and a starting point for drug development.

We developed a new artificial DNA triplex triad which has the adenine nucleobase in the first strand. This triplex triad (A-psi-C*) is composed of adenine, pseudouridine and 5-sudstituted cytosine as the 1st, 2nd and 3rd strand nucleobases, respectively. The molecular design and synthesis of the nucleobases for the 2nd and 3rd strand of the triplex are also described.

Incorporation of nonnatural amino acids into proteins is a useful technique to analyze protein structure and function. We have reported that amber suppressor tRNAs suitable for efficient and specific incorporation of nonnatural amino acids into proteins can be obtained by screening a wide variety of naturally occurring tRNAs in an E. coli. cell-free translation system. The amber suppressor activity of the tRNAs was evaluated by incorporation of a fluorescent nonnatural amino acid and fluorescent SDS-PAGE analysis of cell-free translation products, though the SDS-PAGE was troublesome and time-consuming. In this research, we developed an alternative method for the screening of amber suppressor tRNAs by real-time measurement of fluorescence resonance energy transfer (FRET) from GFP to BODIPY558-linked nonnatural amino acid, which was incorporated into the N-terminus of GFP by amber suppressor tRNAs. Using this method, we demonstrated that the screening of the amber suppressor activity of various prokaryotic Trp tRNAs was performed in a high-throughput manner.

Transfer RNA (guanine-N(2)-)-methyltransferase [tRNA (m(2)G10) methyltransferase] catalyzes a methyl-transfer from S-adenosyl-L-methionine to N(2)-atom of guanine at position 10 (G10) in tRNA and generates N(2)-methylguanine at position 10 (m(2)G10). Yeast enzyme contains two protein subunits (Trm11 and Trm112). Trm11 protein is expected to be a catalytic subunit and Trm112 contains a Zinc-finger. In yeast cells, Trm112 binds not only to Trm11 but also to other proteins such as Lys9, Trm9, and Mtq2. Therefore, the Trm112 protein may regulate population of several protein complexes. To address these issues, we started the study on synthesis of Trm112 related protein complexes. In this meeting, we report synthesis of active Trm11-Trm112 complex in a wheat germ cell-free translation system.