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Cloning of Two Chemically Synthesized Genes for a Precursor to the su+3 Suppressor tRNATyr

Michael J. Ryan, Eugene L. Brown, Ramamoorthy Belagaje, H. Gobind Khorana, Hans-Joachim Fritz


There are three tRNATyr genes in Escherichia coli. One of these genes is found near 88 minutes on the E. coli genetic map; the other two are present as a tandem duplication near the bacteriophage φ80 attachment site at 27 minutes. The structures and organization of these three genes are described in detail by Rossi et al. (this volume). The su+3 amber suppressor tRNATyr has arisen as a result of an anticodon mutation in one of the tandem, duplicate tyr tRNA genes. This su+3 tRNA gene was an ideal goal for the chemical synthesis of a gene because its promoter sequence could be determined (Sekiya et al. 1975Sekiya et al. 1976a,b), it is a relatively small gene, and it has an in vivo function that can be detected easily. This paper describes the cloning and characterization of both the chemically synthesized gene for a precursor to the su+3 tRNA as well as a second, modified derivative of this gene.

Figure 1 represents the total synthetic gene for a precursor to the su+3 tRNA. This synthetic gene includes 56 bp in the promoter region, 126 bp in the structural gene that codes for the su+3 tyr tRNA precursor sequenced previously (Altman and Smith 1971), and 25 bp beyond the mature 3′ end of this tRNA. Originally, it was thought that this region might contain a transcription termination signal. However, recent data have shown that transcription in vitro proceeds for approximately 225 nucleotides before a rho-dependent termination...

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