Satoshi Nishikawa

Construction of a tRNA-embedded-ribozyme trimming plasmid

We have combined Cotten and Birnstiels tRNA-embedded ribozymes and our 5- and 3-trimming system. Although the activity of the tRNA-embedded ribozyme was ca. 30% lower than those of naked ribozymes, since the stability of the former in bovine serum was higher than those of naked ribozymes, the tRNA-embedded ribozymes appear useful especially when the 5- and 3-trimming units are concatenated in tandem.

Systematic substitution of individual bases in two important single-stranded regions of the HDV ribozyme for evaluation of the role of specific bases

To elucidate the role of specific bases in the self‐cleavage activity of the human hepatitis delta virus (HDV) ribozyme, systematic substitutions of individual bases in two important single‐stranded regions [between nucleotides 726–731 (SSrA region) and 762–766 (SSrB region)] were carried out by oligonucleotide‐directed point mutagenesis. Among the mutants obtained, 12 mutants (G726 variants, G727A, G727C, G728C, G762A, G762C, C763 variants and A766C) could not tolerate the respective base‐substitutions and self‐cleavage activities were reduced to very low levels (10%), suggesting a requirement of the respective bases. In particular, G726 in the SSrA region and C763 in the SSrB region were found to be essential for the ribozyme activity. We could determine the preferred sequences, 5′‐G‐G‐(G/A/U)‐N‐(A/U/G)‐Pu‐3′for SSrA and 5′‐(G/U)‐C‐N‐(A/G/U)‐A‐3′ for SSrB regions, respectively.

Point and compensation mutations to evaluate essential stem structures of genomic HDV ribozyme.

In elucidating the part played by the essential stem structures (I, II, and III) in the self‐cleavage activity of genomic HDV ribozyme, several point and compensation variants were constructed on pseudoknot‐like structure by site‐directed mutagenesis. The self‐cleavage activities of these variants indicated that stems I and III were essential for the activity by forming Watson‐Crick base pairs. On the other hand, disruption of A704:U767 had little influence on the cleavage activity, indicating that it is not essential in forming an active structure. Also, our V1 nuclease probing studies showed that the A704U and HDV88 variants have a sensitivity similar to the nuclease, and major cuts are visible in the stem I and stem II regions. Thus, stem I and stem II regions are maintained together with stem III regions in both molecules. These results and our earlier site‐directed mutagenesis studies strongly support a pseudoknot‐like structure for the genomic HDV ribozyme.— Kumar, P. K. R., Suh, Y.‐A., Taira, K., Nishikawa, S. Point and compensation mutations to evaluate essential stem structures of the genomic HDV ribozyme. FASEB J. 7: 124‐129; 1993.

Combination of Trp and Glu residues for recognition of mRNA cap structure : analysis of m7G base recognition site of human cap binding protein (IF-4E) by site-directed mutagenesis

Four mutants of the human cap binding protein (hCBP), in which Trp‐102, Glu‐103, Asp‐104 or Glu‐105 was changed to the aliphatic Leu or Ala, were prepared, and their cap binding abilities were examined. Cap binding abilities of two mutants. W102L (Trp‐102→Leu) and E105A (Glu‐105→Ala), were significantly decreased in comparison with the wild‐type hCBP. This result suggest that Trp‐102 and Glu‐105 are both necessary for the cap binding, and the most probable binding mode with the m7G of cap structure is the combination of the stacking by Trp‐102 and the hydrogen‐bond pairing by Glu‐105, as was already proposed from the model studies.

Constructing an efficient trans‐acting genomic HDV ribozyme

We have engineered a genomic HDV ribozyme to construct several trans‐acting ribozymes for use in trans to cleave target RNAs. Among the 10 different combinations attempted, only HDV88‐Trans had cleavage activity on the 13‐nucleotide substrate, R13, in vitro. To improve the cleavage efficiency, at least in vitro, of the HDV88‐Trans ribozyme (kclv = 0.022 min−1), we have constructed several variants that differ in forming stem II (length) in the pseudoknot secondary structure model. When cleavage rate constants were analyzed and compared among variants of HDV88‐Trans, HDV88‐Trans‐4 yielded kclv = 1.7 min−1. HDV88‐Trans‐4 thus represents the highest active genomic HDV ribozyme that functions in trans thus far constructed, and has activity under physiological conditions (pH 7.1 at 37°C with 1 mM of MgCl2).

PURIFICATION OF T4 RNA LIGASE BY 2',5'-ADP SEPHAROSE CHROMATOGRAPHY

RNA hgase was discovered in T4 infected Escherichia coli cells [ 11. This enzyme catalyzes the intramolecular loinmg of 5’.P and 3’.OH termini of polynucleotides and the intermolecular joining of various combmations of oligoand polynucleotides at their 5’.P and 3’.OH termmi [2-81. RNA ligase also catalyzes the addition of dinucleoside pyrophosphates [9] and mononucleotides [lO,l l] to oligonucleotide acceptors. Nucleoside 3’,5’-diphosphates are the shortest donor substrates and their 3’.phosphates are essential for the reaction because nucleoside S’-phosphates and nucleoside 2’,5’-drphosphates are unable to serve as donors [9-l 11. We found that 2’,5’-ADP inhibited the RNA hgase reaction. The procedures for punfication of RNA ligase have been improved by several groups [2,3,5,12,13]. However, it was sometrmes difficult to remove a trace of nuclease activity from RNA ligase preparations. Recently, we found that RNA hgase bound to 2’S’ADP Sepharosein the presence of Mg2* but not in its absence, and we introduced the 2’S’-ADP Sepharose chromatography at the last step of our purification procedure. The enzyme so obtained had no detectable RNase activity and was suitable for the synthesis of oligoribonucleotides with defined sequences.

Secretion of recombinant ribonuclease T1 into the periplasmic space of Escherichia coli with the aid of the signal peptide of alkaline phosphatase

The ribonuclease T1 (RNase T1) gene was ligated to a synthetic gene for the signal peptide of Escherichia coli alkaline phosphatase. When this fusion gene was expressed in E.Coli under the control of the trp promoter, active RNase T1 having the correct N‐terminal sequence was secreted into the periplasmic space, indicating that the heterologous signal peptide had been cleaved off correctly. The enzyme could be readily purified from the periplasmic fraction with a yield of 1.8 mg from 1 liter culture. Adopting the same strategy, it was possible to produce a labile mutant of RNase T1 (Glu‐58 → Ala mutant) in E. coli, the yield of the purified mutant enzyme being 2.0 mg from 1 liter culture.

RNA hydrolysis via an oxyphosphorane intermediate

From calculations of a model reaction scheme for base-catalyzed RNA hydrolysis, a pentacoodinate dianionic intermediate 2a (Storer, et al., J. Am. Chem. Soc., 1991, 113, 5216-5219) as well as two transition states, TS1 and TS2, to the intermediate have been located by ab initio calculations at the 3-21G* level. Although the intermediate, which has the well depth on the order of kBT, is unlikely to be kinetically significant, the overall rate-limiting transition state structure TS2 obtained at 3-21G* level is very close to the corresponding structure at the STO-3G level; it has an extended P-O(5) bond breaking character. These gas-phase calculation results are used to qualitatively interpret mutagenesis results of Barnase and RNase T1 where water molecules are absent from the active site.

Mechanism of hydrolysis of phosphodiesters with ribonuclease T1

Ribonuclease Tl (RNase Tl) specifically hydrolyzed the phosphodiester linkages of guanosine 3-phosphate of single—stranded RNA and the cleavage occurs in a two—step reaction mechanism. It is considered that in the first step Glu 58 abstracts a proton from 2-OH and His 40 or His 92 adds a proton to 0-5 of ribose. We succeeded to express the chemically synthesized genes for RNase Tl and its several mutants at base-recognition site in E. coli and reported the structure-function relationship of this enzyme. In this paper, we changed Glu 58, His 40 and His 92 to alanine etc. and analyzed the activity of these mutant enzymes in order to clarify the role of catalytic residues. Gin 58 mutant and Ala 58 mutant still retained slight activity but both Ala 40 and Ala 92 mutants lost the activity almost completely. This result indicates that Glu 58 is not essential but His 40 and His 92 are indispensable for RNase Ti activity. We propose a new reaction mechanism, in which His 40 abstracts a proton from 2-OH and His 92 protonates 0-5 of ribose while Glu 58 enhances the basicity of His 40.

Polynucleotides: XXVIII. Stimulation of the binding of aminoacyl-tRNA to ribosomes by tri- and polynucleotide analogs

Messenger activity of synthetic tri- and polynucleotide analogs was studied by binding of 14C-labeled aminoacyl-tRNAs to ribosomes in the presence of the analogs. Synthetic messengers used were: poly(A) analogs in which adenosine was replaced by tubercidine (I), 3-deazaadenosine (II), 1-deazaadenosine (III) and 2-methyladenosine (IV); copolymers of adenosine and aristeromycin (V); cyclic triadenylate (VI); the heptanucleotide of 6,2-O-cyclouridine (VII); the pentanucleotide of 8,2-S-cycloadenosine (VIIIa); A-U-G analogs in which adenosine was replaced by 8,2-O- and S-cycloadenosine (VIII), 8,5-O- and S-cycloadenosine (IX); 8-oxyadenosine (x); 8-bromoadenosine (XI) and formycine (XII). Among these oligo- and polynucleotides, analogs which contained nucleotides of anti conformation having appropriate bases for Watson-Crick type hydrogen bonding stimulated the binding of corresponding tRNAs to ribosomes.