Transcriptional analysis of puf operon expression in Rhodobacter sphaeroides 2.4.1 and an intercistronic transcription terminator mutant

DNA sequence analysis of the pufX region, the most distal gene of the pufBALMX operon of Rhodobacter sphaeroides, revealed a sequence encoding a putative polypeptide of 82 amino acids with a molecular mass of 9052 Da followed by a puf operon-specific transcription terminator. Analysis of the 5′ and 3′ termini of the transcripts produced in vivo from the puf operon of R. sphaeroides PUF Δ 348–420 (three transcripts; 0.59, 0.64, and 2.63 kilobases) lacking the puf-intercistronic terminator structure were identical to those of the corresponding puf transcripts derived from wild-type R. sphaeroides 2.4.1 (four transcripts; 0.50, 0.66, 0.71, and 2.7 kilobases) showing that the transcripts begin and end at the same sites. However, the absence of the puf intercistronic terminator resulted in both the loss of the smallest transcript found in wild type and increased transcriptional read-through of the mutated region to the more distal pufL gene, supporting our previous contention that the proximal intercistronic stem-loop functions as a transcription terminator. The 5′ terminus of the medium sized puf transcript has been localized to the same site as that of the small puf transcript. These analyses also showed conclusively that the puf operon-specific transcripts are not extended transcripts derived from the upstream open reading frame Q. In addition, a 120-nucleotide RNA was detected which encompassed the terminator region downstream of pufX and extended into the next downstream open reading frame. The 120-nucleotide RNA of unknown function was regulated by O2 and is unique in its abundance and stability. By comparison with strain 2.4.1, the mutant PUF Δ 348–420 showed an increased amount (1.9-fold) of the 120-nucleotide RNA, suggesting that its synthesis is under the control of the puf operon despite the fact that its sequence appears to overlap the next downstream operon.