Phylogenetic conservation of RNA secondary and tertiary structure in the trpEDCFBA operon leader transcript in Bacillus.
Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms. We recently determined that the B. subtilis trp leader readthrough transcript can adopt a Mg(2+)-dependent tertiary structure that appears to interfere with TRAP-mediated translation control of trpE. In the present study, sequence comparisons to trp leaders from three other Bacillus sp. were made, suggesting that RNA secondary and tertiary structures are phylogenetically conserved. To test this hypothesis, experiments were carried out with the trp leader transcript from Bacillus stearothermophilus. Structure mapping experiments confirmed the predicted secondary structure. Native gel experiments identified a faster mobility species in the presence of Mg(2+), suggesting that a Mg(2+)-dependent tertiary structure forms. Mg(2+)-dependent protection of residues within the first five triplet repeats of the TRAP binding target and a pyrimidine-rich internal loop were observed, consistent with tertiary structure formation between these regions. Structure mapping in the presence of a competitor DNA oligonucleotide allowed the interacting partners to be identified as a single-stranded portion of the purine-rich TRAP binding target and the large downstream pyrimidine-rich internal loop. Thermal denaturation experiments revealed a Mg(2+)- and pH-dependent unfolding transition that was absent for a transcript missing the first five triplet repeats. The stability of several mutant transcripts allowed a large portion of the base-pairing register for the tertiary interaction to be determined. These data indicate that RNA secondary and tertiary structures involved in TRAP-mediated translation control are conserved in at least four Bacillus species.
Schaak
Janell E
JE
Department of Biochemistry and Molecular Biology, The Huck Institute for Life Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Babitzke
Paul
P
Bevilacqua
Philip C
PC
D001483:Base Sequence; D001411:Geobacillus stearothermophilus / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009876:Operon; D010802:Phylogeny; D012329:RNA, Bacterial / Q000737:chemistry*; D012333:RNA, Messenger / Q000737:chemistry*
D012329:RNA, Bacterial; D012333:RNA, Messenger