The nucleotide sequence in the promoter region of the fene for an Escherichia coli tyrosine transfer ribonucleic acid

Abstract

The sequence of 29 nucleotides in the region preceding the initiation of the transcription of the Escherichia coli tyrosine tRNA gene has been determined. This is: (5') ---GGGGCGCATCATATCAAATGACGCGCCGC--- (3') (3') ---CCCCGCGTAGTATAGTTTACTGCGGCGGCG--- (5'). The general approach used for the sequence determination involved the DNA polymerase I-catalyzed elongation of suitable deoxyribopolynucleotide primers when hybridized to the 1-strand of phi80psuIII plus DNA at the appropriate site. Sequences of the newly grown oligonucleotide chains were determined by a combination of two-dimensional fingerprinting following partial exonucleolytic degradation, nearst neighbor analyses, and determination of pyrimidine tracts. Primer elongations were carried out in a controlled and stepwise manner and the newly grown oligonucleotide chains were kept short by incorporating the following features into the method: (a) the insertion of a ribonucleotide unit at or near the 3' terminus of the primers; (b) the use of a maximum of three nucleoside 5'-triphosphates in the first stage of the elongation reaction isolation of the elongated primer, and its reuse in a second step together with different sets of deoxy-nucleoside triphosphates; and (c) elongation of the primer using all of the four nucleoside triphosphates with one of the triphosphates being supplied in a limiting concentration.