Mapping of the puh messenger RNAs from Rhodospirillum rubrum. Evidence for tandem promoters

Abstract

The mRNA transcripts of Rhodospirillum rubrum gene puh, coding for the H subunit of the photoreaction center, and of genes flanking puh were analyzed by blot hybridization. Open reading frame G115, upstream of structural gene puh, is transcribed as a 2.25-kilobase mRNA. Gene puh itself is transcribed as two mRNAs of 1118 and 1032 nucleotides. Mung bean nuclease protection analysis shows that the puh transcripts have different 5' termini within open reading frame G115 and a unique rho-independent termination signal within open reading frame I2372. The lifetimes of the puh messages, as determined by an oxygen blockade of transcription, were 10 and 12 min for the large and small puh mRNAs, respectively. An expression vector carrying a chloramphenicol acetyltransferase gene was used to select promoters in DNA stretches upstream of the startpoints of each of these transcripts. Chloramphenicol resistance was expressed in Escherichia coli, using as a promoter a 179-nucleotide stretch upstream of the small mRNA startpoint but not from a 124-nucleotide stretch upstream of the large mRNA startpoint. The promoter for the small mRNA, designated Ppuh2, is thought to encompass in its -10 and -35 regions a sigma 70-like RNA polymerase recognition sequence. The region upstream of the large message startpoint contains a sequence similar in its -12 and -24 regions to promoter sequences recognized by the sigma 60 RNA polymerase holoenzyme. This is designated as promoter Ppuh1.Ppuh1 is proposed to be strictly regulated by light intensity and by oxygen tension while Ppuh2 would be less sensitive to these parameters.