The complete nucleotide sequence of the 23S ribosomal RNA gene from the rrnB operon of Escherichia coli has been determined. The sequences of both strands of the entire gene were determined, and most of the sequence was independently confirmed by use of alternate restriction fragments. The DNA region corresponding to mature 23S rRNA contains 2904 nucleotides. Kethoxal-reactive sites protected by 30S subunits are found between positions 2300 and 2800, placing the subunit interface in this region of the molecule. The functional importance of this region is further supported by studies by other investigators, including homology with chloroplast and mitochondrial rRNA. Elucidation of the structure of the ribosome poses a major challenge for molecular biologists. Deduction of the primary structures of the rRNAs is a necessary prelude to solving their three-dimensional structures, as well as to understanding the complex series of events that take place during ribosome assembly, the molecular mechanisms underlying protein synthesis, and the molecular evolution of the translational machinery. Primary structures for Escherichia coli 5S rRNA and 16S rRNA have been completed, and this study reports the DNA sequence of the 23S rRNA gene from the rrnB cistron of E. coli. This completes the primary structural analysis of the E. coli rRNAs. The entire rrnB 23S rRNA gene is contained in the 2.15-kb insert of pER18 and the 3.2-kb insert of pKK123. The restriction fragments used in deducing the sequence are shown schematically in Fig. 1. The sequences of the rrnB spacer regions flanking the 23S RNA and 5S RNA genes will be published elsewhere. The region included in pER18 could be sequenced, overlapped, and confirmed by use of fragments obtained by digestion with Ava II, HinfI, Hpa II, Hph I, Mbo II, and Taq I. The pKK123 insert was digested with Hpa II, BstNI, Hph I, and, in combination, Ava I/Taq I and HinfI/Sau96I. Both strands of the entire gene have been sequenced, and most of the sequence has been reconfirmed by use of overlapping restriction fragments. The entire sequence was done by the chemical method of Maxam and Gilbert. Sequences extending 200–300 nucleotides from the labeled end could be routinely and completely read. The complete rrnB 23S RNA sequence is shown in Fig. 2. Placement of post-transcriptionally modified nucleotides as well as the 5' and 3' termini is based on results from other laboratoriesThe complete nucleotide sequence of the 23S ribosomal RNA gene from the rrnB operon of Escherichia coli has been determined. The sequences of both strands of the entire gene were determined, and most of the sequence was independently confirmed by use of alternate restriction fragments. The DNA region corresponding to mature 23S rRNA contains 2904 nucleotides. Kethoxal-reactive sites protected by 30S subunits are found between positions 2300 and 2800, placing the subunit interface in this region of the molecule. The functional importance of this region is further supported by studies by other investigators, including homology with chloroplast and mitochondrial rRNA. Elucidation of the structure of the ribosome poses a major challenge for molecular biologists. Deduction of the primary structures of the rRNAs is a necessary prelude to solving their three-dimensional structures, as well as to understanding the complex series of events that take place during ribosome assembly, the molecular mechanisms underlying protein synthesis, and the molecular evolution of the translational machinery. Primary structures for Escherichia coli 5S rRNA and 16S rRNA have been completed, and this study reports the DNA sequence of the 23S rRNA gene from the rrnB cistron of E. coli. This completes the primary structural analysis of the E. coli rRNAs. The entire rrnB 23S rRNA gene is contained in the 2.15-kb insert of pER18 and the 3.2-kb insert of pKK123. The restriction fragments used in deducing the sequence are shown schematically in Fig. 1. The sequences of the rrnB spacer regions flanking the 23S RNA and 5S RNA genes will be published elsewhere. The region included in pER18 could be sequenced, overlapped, and confirmed by use of fragments obtained by digestion with Ava II, HinfI, Hpa II, Hph I, Mbo II, and Taq I. The pKK123 insert was digested with Hpa II, BstNI, Hph I, and, in combination, Ava I/Taq I and HinfI/Sau96I. Both strands of the entire gene have been sequenced, and most of the sequence has been reconfirmed by use of overlapping restriction fragments. The entire sequence was done by the chemical method of Maxam and Gilbert. Sequences extending 200–300 nucleotides from the labeled end could be routinely and completely read. The complete rrnB 23S RNA sequence is shown in Fig. 2. Placement of post-transcriptionally modified nucleotides as well as the 5' and 3' termini is based on results from other laboratories