Terumichi Tanaka

Extracellular DNA and RNA produced by a marine photosynthetic bacterium Rhodovulum sulfidophilum

A marine photosynthetic bacterium Rhodovulum sulfidophilum secretes nucleic acids that are involved in flocculating ability. These extracellular nucleic acids have not been well characterized. Here, we have analyzed these nucleic acids and revealed that the extracellular nucleic acids are a mixture of double-stranded DNAs and single-stranded RNAs. The DNAs have sizes of more than 30 kbp and at least a part of these DNAs is probably an amplified portion of genomic DNA. The RNAs seems to be tRNA like molecules from size estimation.

Guide DNA technique reveals that the protein component of bacterial ribonuclease P is a modifier for substrate recognition.

We developed a guide DNA technique with which the cleavage efficiency of pre‐tRNA substrate raised in the RNase P reaction. The 20‐mer guide DNAs hybridizing to the upstream region of the cleaving site enhanced the cleavage reactions of RNA substrates by Escherichia coli RNase P. This guide DNA technique was also applicable to cleavage site selection by choosing the DNA‐hybridizing site. Results showed that RNase P accepts DNA/RNA double‐stranded 5′‐leader region with high catalytic efficiency as well as single‐stranded RNA region in pre‐tRNAs as substrates, which suggests that the protein component of bacterial RNase P prefers bulky nucleotides. The protein component did not affect the normal 5′‐processing reaction of pre‐tRNAs, but enhanced the mis‐cleaving (hyperprocessing) reactions of tRNA in non‐cloverleaf folding. Our results suggested that the protein component of RNase P is a modifier for substrate recognition.

Artificial RNA aptamer production by the marine bacterium Rhodovulum sulfidophilum: Improvement of the aptamer yield using a mutated transcriptional promoter

Noncoding small RNAs and artificial RNA aptamers are now expected to be potential candidates for RNA therapeutic agents. We previously proposed a unique method for economical production of these RNAs using the marine phototrophic bacterium Rhodovulum sulfidophilum. This bacterium does not produce any ribonucleases but does produce extracellular nucleic acids in the culture medium in nature. Using this bacterium and an engineered plasmid containing the rrn promoter for the RNA expression, we developed a method for production of the streptavidin RNA aptamer in the culture medium. However, the yield of this RNA product in the culture medium by this method was not enough for practical use. In the present paper, we improved the yield of this product by modification of the -35 region of the rrn promoter so as to escape from the Fis protein control and the use of a new vector plasmid. Using this system, the extracellular RNA aptamer of approximately 200 ng and the total RNA aptamer (both extra- and intracellular form) of about 20 μg from 1 L culture were accomplished by constitutive expression of the gene.

Examining the bases of the J3/4 domain of Escherichia coli ribonuclease P.

We prepared several mutants of the J3⁄4 and P4 domains of Escherichia coli ribonuclease P (RNase P): A62G, A62U, G63C/G64C, A65G, A67G, U69A, U69G, U69C, U69Δ, and U69UU. Comparison of the ribozyme and holo enzyme reactions at various concentrations of magnesium ions showed that the presence of a bulge at U69 in the P4 domain was important in the holo enzyme. The results also showed that the conserved bases G63 and G64 in the J3⁄4 domain were important for efficient ribozyme reactions but were replaceable in the presence of the protein component. Our data showed that the bases in the J3⁄4 and P4 domains displayed different responses to the metal ions that were affected by the presence of the protein component.

Mutational analysis of the length of the J3/4 domain of Escherichia coli ribonuclease P ribozyme

We prepared a series of length variants of the J3/4 domain of Escherichia coli ribonuclease P (RNase P) ribozyme: the four-base long J3/4 domain (A62G63G64A65) was replaced with GGA (denoted ΔA), GA (ΔAG), A (ΔAGG), AAGGA (ΣA), AAAGGA (ΣAA), and AAAAGGA (ΣAAA). The results indicated that truncating and inserting operations of the J3/4 domain drastically reduced ribozyme activity (WT>>ΣAA>ΣA>ΣAAA>>ΔAG>ΔA, ΔAGG), but did not affect the cleavage site selection of a substrate by the ribozyme. The reduced ribozyme activity of each mutant was rescued to some extent by the addition of a high concentration of magnesium ions. Our data indicate that the conserved AGGA sequence was important for efficient ribozyme reactions, and suggested that the length mutations affected ribozyme activity through metal ion binding steps.

The catalytic RNA of RNase P from Escherichia coli cleaves Drosophila 2S ribosomal RNA in vitro: A new type of naturally occurring substrate for the ribozyme

We have found that the catalytic RNA of RNase P of Escherichia coli (M1 RNA) can cleave 2S ribosomal RNA (2S rRNA) of Drosophila melanogaster at specific positions in vitro. The cleavage mainly occurred at two sites between nucleotides 11 and 12, and between 16 and 17 of 2S rRNA. Kinetic analyses of the reaction revealed that a dimer caused by intermolecular interaction of 2S rRNA may be the substrate for the cleavage between 11 and 12, while a simple monomer is the substrate for the cleavage between 16 and 17. Substrate recognition by M1 RNA is also discussed.

High-affinity RNA aptamers to C-reactive protein (CRP): newly developed pre-elution methods for aptamer selection

We have developed a modified SELEX (systematic evolution of ligands by exponential enrichment) method to obtain RNA aptamers with high affinity to C-reactive protein (CRP). CRP is a clinical biomarker present in plasma, the level of which increases in response to infections and noninfectious inflammation. The CRP level is also an important prognostic indicator in patients with several syndromes. At present, CRP content in blood is measured immunochemically using antibodies. To develop a more sensitive method using RNA aptamers, we have attempted to obtain high-affinity RNA aptamers to CRP. We succeeded in obtaining an RNA aptamer with high affinity to CRP using a CRP-immobilized Sepharose column and pre-elution procedure. Pre-elution is a method that removes the weak binding portion from a selected RNA population by washing for a short time with buffer containing CRP. By surface plasmon-resonance (SPR) analysis, the affinity constant of this aptamer for CRP was calculated to be KD = 2.25?10?9 (M). The secondary structure, contact sites with CRP protein, and application of this aptamer will be described.

The P3 domain of E. coli ribonuclease P RNA can be truncated and replaced

We prepared some truncated and replaced P3 mutants of Escherichia coli RNase P RNA, and used them to examine the RNase P ribozyme and holoenzyme reactions of a pre‐tRNA substrate. The results indicated that mutations in the P3 domain did not affect the cleavage site selection of the pre‐tRNA substrate, but did affect the efficiency of cleavage of the substrate. Results of stepwise truncation of the P3 domain and its replacement by the TAR sequence showed that the P3 domain of the E. coli RNase P was able to be truncated to certain length and was replaceable, but could not be deleted in the ribozyme.

The Protein Component of Bacterial Ribonuclease P Flickers the Metal Ion Response to the Substrate Shape Preference of the Ribozyme

The substrate shape specificity of the Escherichia coli ribonuclease P (RNase P) ribozyme depends on the concentration of magnesium ion. At 10 mM or more, it can cleave a hairpin substrate as well as a cloverleaf pre-transfer RNA (tRNA). The results showed, however, that the holo enzyme cleaved the hairpin substrate at low concentrations of magnesium ion. Considering that the homologous E. coli tRNAs are resistant to internal cleavage by the RNase P, the phenomena suggest that this catalytic activity might take part in the removing the mis-folded RNAs in the cell.

Guide DNA technique in bacterial ribonuclease P reaction for effective processing of tRNA precursor

Previously, we found that a small (approx. 20‐mer) DNA hybridizing to the 5′‐leader region of a tRNA precursor enhances the cleavage efficiency in bacterial ribonuclease P reaction. We named this technique the ‘guide DNA technique’. Detailed analyses showed that the length of the guide DNA, concentration of the guide DNA and the hybridizing position affected the cleavage efficiency: for an effective cleavage reaction, guide DNA should be designed to hybridize to the region on the cleavage site, should be 20 bases or more in length and should be of high concentration. The presence of a 5′‐flanking region in the DNA did not affect the cleavage reaction. The guide DNA technique is a useful tool for effective preparation of mature tRNA molecules in vitro.