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.

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An unnatural base pair of 2-amino-6-(N,N-dimethylamino)purine (designated as x) and pyridin-2-one (designated as y) has been developed for specific transcription. The ribonucleoside triphosphates of y and a modified y, 5-methylpyridin-2-one, are selectively incorporated into RNA opposite x in the templates by T7 RNA polymerase. In addition, the sequences of the DNA templates containing x can be confirmed by a dideoxynucleotide chainterminator method supplemented with the deoxynucleoside triphosphate of y. The bulky dimethylamino group of x in the templates effectively eliminates noncognate pairing with the natural bases. These results enable RNA biosynthesis for the specific incorporation of unnatural nucleotides at the desired positions.T ranscription involving specific, unnatural base pairs, in addition to the A⅐U and G⅐C pairs, would be a method for the site-directed incorporation of unnatural bases into RNAs, to achieve new functionality as ligands or catalysts (1-5) or novel codon-anticodon interactions between transfer and messenger RNAs in translation (6-9). Compared with the chemical synthesis of RNA, transcription is more efficient to produce and amplify RNAs with long chain lengths from DNA templates, which can be constructed by chemical synthesis in combination with enzymatic ligation. Thus, many efforts have been made to develop unnatural bases that are recognized by RNA polymerases as substrates and are specifically incorporated into RNA opposite the pairing bases in the DNA templates.Previous efforts to create unnatural base pairs for replication and transcription have relied on nonstandard hydrogen-bonding schemes that differ from those of the Watson-Crick base pairs (2, 10 -16). Unnatural pairs of nucleotides, such as isoguanosine⅐isocytidine and xanthosine⅐2,4-diaminopyrimidine nucleoside, are complementarily incorporated into DNA and RNA by polymerases with moderate selectivity (11-15). In addition, in vitro translation for the site-specific incorporation of an unnatural amino acid into a peptide has been demonstrated by using an extra codon-anticodon interaction between isoguanine and isocytosine (6, 7). These studies indicate that the unnatural, nonstandard hydrogen-bonded base pairs can also function in replication and transcription. However, some misincorporations resulting in noncognate pairings with natural bases cannot be ignored for the practical usage of these unnatural base pairs. For instance, a 14% misincorporation of adenosine into RNA opposite 2,4-diaminopyrimidine is observed at a xanthosine triphosphate-to-ATP ratio of 1:1 in transcription by T7 RNA polymerase (2). Isoguanine tautomerization also causes the misincorporation of T or U opposite this base (11,12). In addition, the isocytidine and 5-methylisocytidine nucleoside derivatives are chemically unstable, and gradually decompose in solution (11,16). Furthermore, nucleoside triphosphates of 2-aminopyrimidines, such as isocytosine and 2,4-diaminopyrimidine, are not recognized as substrates by some polymerases (11,14,15) becau...