Activity and thermostability of the small self-splicing group I intron in the pre-tRNA(lle) of the purple bacterium Azoarcus.
The 205-nt group I intron located in the pre-tRNA(lle) from the bacterium Azoarcus sp.BH72 is the smallest self-splicing group I intron identified to date. Comparative sequence analysis has placed this intron and the Anabaena pre-tRNA(Leu) intron into the same subgroup, IC3; we now compare their activity and stability. Unlike the Anabaena intron, the Azoarcus intron has two transitions in the kinetics of the first step of splicing. The faster transition occurs with a larger k(cat)/K(m) than that of the Anabaena or other group I introns, due to a rapid K(cat) (5 min(-1) at 32 degrees C) and a low K(m) for guanosine (17 microM). The excised intron circularizes by releasing a trinucleotide from the 5' end of the intron, another property unlike the Anabaena intron. Although it is smaller in size, the Azoarcus intron retains activity at higher temperatures, higher concentrations of urea, and higher pH than the Anabaena intron. Melting curves show that tertiary structure is disrupted at a lower temperature in the Anabaena intron. Some structural features that may explain the unusual stability of the Azoarcus intron include a G-C rich secondary structure and the presence of two 11-nt motifs, which are known to interact strongly with GAAA loops in group I and group II introns. The disruption of one of these interactions by substituting the Anabaena structural element in fact lowered the thermal stability of the Azoarcus intron. Thus, even superficially similar group I introns from the same structural subgroup can differ significantly in activity and stability.
Tanner
M
M
Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder 80309-0215, USA.
Cech
T
T
D001483:Base Sequence; D006092:Gram-Negative Facultatively Anaerobic Rods / Q000235:genetics*; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D012355:RNA, Transfer, Ile / Q000235:genetics* / Q000378:metabolism; D013696:Temperature
D012322:RNA Precursors; D012355:RNA, Transfer, Ile