M. Ikehara

Photo CIDNP study on the complex formation of λ- cro protein with OR3

A photo CIDNP spectrum of λ cro repressor protein showed that one of the three tyrosines and His 35 are quite accessible to the photosensitive dye. For the remaining two tyrosine residues one is slightly accessible, but the other is inaccessible. In comparison with the result of differential nitration at tyrosine side chains followed by the peptide analysis, it can be concluded that Tyr 26 is mostly exposed and Tyr 51 is slightly exposed on the surface of the cro dimer. On the addition of OR3 17mer, His 35 and Tyr 26 are no longer accessible to the dye, which indicates that they are involved in interaction. However, a similar phenomenon was observed by adding CAP binding site 22mer. The interaction mechanism will be discussed.

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.

A----Z transition in the synthetic hexanucleotide (dCdGfl)3.

500 MHz proton NMR and NOE measurements on (dCdGfl)3 show that at very low ionic strength the hexanucleotide adopts an A‐DNA conformation, whereas at high salt concentrations a Z‐form is found. At intermediate salt concentrations the two species are in slow exchange on the proton NMR time scale. This transition was also observed by characteristic changes in the CD spectra.

Crystal and molecular structure of a cyclonucleoside, 8,5′-anhydro-2′,3′-isopropylidene-8-mercaptoadenosine

Abstract The crystal and molecular structure of 8,5′-anhydro-2′, 3′-isopropylidene-8-mercaptoadenosine have been determined by X-ray diffraction methods. The relationship between the sugarbase torsion angle and sign and magnitude of the Cotton effect in purine cyclonucleosides is to be further considered.

Conformational difference between 8-bromo-2′-O-triisopropylbenzenesulfonyl-adenosine and its 3′-isomer determined by x-ray method

Summary The crystal structures of 8-bromo-2′-O-triisopropylbenzenesulfonyl-adenosine and its 3′-isomer have been determined by X-ray diffraction method. In the former crystal, an intra-molecular stacked conformation between adenine- and benzene-ring was found and there exist two modes of dimer formation: an inter-molecular stacked dimer and a hydrogen-bonded one. On the other hand, the 3′-isomer, 8-bromo-3′-O-triisopropylbenzenesulfonyladenosine, has a similar elongated non-stacked conformation as puromycin.

GLU 46 of ribonuclease T1 is an essential residue for the recognition of guanine base

The Glu 46 of ribonuclease T1, which is assumed to interact with Nl of the guanine residue in RNA by a hydrogen bond from the result of X-ray analysis, was changed to alanine by site-directed mutagenesis and its function examined. The nucleolytic activity of the Ala 46 mutant enzyme against pGpC decreased to 0.4% of that of the wild-type enzyme, on the other hand its activity against pApC increased. This result suggests that the Glu 46 is essential for the recognition of the guanine base but that it also interferes with the recognition of the adenine base.

SYNTHESIS OF tRNAfMet FROM E. COLI

Abstract A synthetic approach to the formylmethionine-tRNA from E. coli is described. The methods employed include 1) phosphodiester synthesis using block condensation or stepwise addition methods, 2) a mixed method combining di- and triester approaches, 3) the phosphotriester method using o-nitrobenzyl for 2′-OH, p-chlorophenyl and anilidate for phosphate and acyl groups for base protection, and 4) joining of the synthetic fragments with RNA ligase.