Poster abstracts

Poster number 11 submitted by Chuck Daniels

The tRNA intron endonuclease from the haloarchaeal organism, Haloferax volcanii, plays a broad role in RNA metabolism.

Richard N. Nist (Department of Microbiology, The Ohio State University), Anice Sabag-Daigle (Department of Microbiology, The Ohio State University), Charles J. Daniels (Department of Microbiology, The Ohio State University)

Abstract:
The tRNA intron endonuclease from the haloarchaeal organism, Haloferax volcanii, recognizes its substrate using a structure-specific element, the bulge-helix-bulge motif (BHB). In this motif the cleavage sites are located in two three-nucleotide bulge loops that are separated by four base pairs. This enzyme does not require the presence of mature tRNA structures, a property that distinguishes it from its eukaryal counterparts. The lack of a requirement for mature tRNA structure predicts that the archaeal enzyme could act on molecules other than intron-containing tRNAs and have a broader role in RNA metabolism in this organism. Sequence analysis of the H. volcanii genome revealed three intron-containing tRNAs: tRNATrp, tRNAMet and tRNAGln; each possesses the characteristic BHB motif. We also noted that BHB motifs were present in the helices formed by the interactions of the spacer regions flanking the 16S and 23S rRNA in the predicted rRNA primary transcript. These regions are analogous to the RNase III sites in bacterial rRNA primary transcripts and suggested that the tRNA intron endonuclease may function in the maturation of the rRNA primary transcript in H. volcanii. To address this question we have examined the consequence of over-expressing a catalytically inactive form of the H. volcanii tRNA intron endonuclease protein, EndA*, in vivo using a newly developed plasmid expression system. Expression of the EndA* mutant protein leads to an accumulation of intron-containing pretRNAs indicating the mutant has a dominant effect and that these cells are deficient in endonuclease activity. Northern analysis probing RNAs from the rRNA operon indicate that several intermediates are affected by EndA* expression; one notable example is the presence of 16S-tRNAAla intermediates resulting from decreased cleavage at predicted sites flanking the 16S rRNA. Unexpectedly, we also observed that a small RNA resulting from a cleavage-ligation reaction of sequences flanking the 23S rRNA was absent when EndA* was expressed. This suggests that the tRNA intron splicing system (endonuclease + ligase) is responsible for the formation of a novel spliced RNA from the rRNA primary transcript.

Keywords: Intron processing, tRNA metabolism, Archaea