Preparation of eukaryotic mRNA having differently methylated adenosine at the 5'-terminus and the effect of the methyl group in translation.

Cap analogues having differently methylated adenosine at 2' and N6 position, m(7)G(5')pppApG which is existed in plant mRNA (plant type), m(7)G(5')pppAmpG (animal type), m(7)G(5')pppm(6)AmpG (mammalian type) and m(7)G(5')pppm(6)ApG (unnnatural type), were synthesized. In order to clarify the function of these methyl groups, luciferase mRNAs having differently methylated adenosine at the 5'-terminus, were successfully prepared by in vitro transcription using the synthesized cap anologues. As the preliminary results of in vitro translation with rabbit reticulocyte lysate and luciferase assay, luciferase mRNA having the mammalian type of cap structure, m(7)G(5')pppm(6)AmpG, was most efficiently translated. In the case of m(7)G(5')pppApG (plant type) efficiency of translation was lowest.

Important sequence for overexpression of NADH oxidase gene from Thermus thermophilus HB8 in Escherichia coli.

Enzymes fixed on the electrode of biosensor are gradually inactivated and the electrode is discarded after using several times. In order to prepare the stable biosensor, we try to use a stable enzyme from extreme thermophilic bacteria, Thermus thermophilus HB8. It is very important that a stable enzyme from T. thermophilus HB8 is overproduced in Escherichia coli, for the purpose of enough supply of enzyme. Thereby, we determined the important sequence for overexpression of NADH oxidase (nox) gene from T. thermophilus HB8 in E. coli. As a result, it is revealed that ten nucleotides sequence, GAAATTAACT, in the upstream of start codon of nox gene was important for its overexpression in E. coli.

A unique unnatural base pair between a C analogue, pseudoisocytosine, and an A analogue, 6-methoxypurine, in replication.

Pseudoisocytidine, a C-nucleoside analogue of cytosine, has two possible isomers of the H1- and H3-forms. Enzymatic incorporation experiments confirmed the existence of the two isomers in solution, and the 2'-deoxyribonucleoside triphosphate of pseudoisocytosine (PIC) was incorporated into DNA opposite both guanine and 6-methoxypurine (M) by the Klenow fragment of Escherichia coli DNA polymerase I. In addition to the PIC*M pairing in replication, M also functioned as an A analogue and T was efficiently incorporated opposite M. Thus, the PIC*M pair is regarded as a base pair between a C analogue and an A analogue, and can mediate the interconversion between the G*C and A*T base pairs. The combination of PIC and M could be used as a G*C<-->A*T transition mutagen.

Crystal structures of 7-methylguanosine 5'-triphosphate (m(7)GTP)- and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)-bound human full-length eukaryotic initiation factor 4E: biological importance of the C-terminal flexible region.

The crystal structures of the full-length human eukaryotic initiation factor (eIF) 4E complexed with two mRNA cap analogues [7-methylguanosine 5'-triphosphate (m(7)GTP) and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)] were determined at 2.0 A resolution (where 1 A=0.1 nm). The flexibility of the C-terminal loop region of eIF4E complexed with m(7)GTP was significantly reduced when complexed with m(7)GpppA, suggesting the importance of the second nucleotide in the mRNA cap structure for the biological function of eIF4E, especially the fixation and orientation of the C-terminal loop region, including the eIF4E phosphorylation residue. The present results provide the structural basis for the biological function of both N- and C-terminal mobile regions of eIF4E in translation initiation, especially the regulatory function through the switch-on/off of eIF4E-binding protein-eIF4E phosphorylation.