Poster: Organelle biogenesis

Abs # 634: A group II intron from the chloroplast psbA gene of a psychrophilic Chlamydomonas: In vitro self-splicing and genetic evidence for maturase activity

Presenter:	Herrin, David L., herrin@mail.utexas.edu
Authors	Herrin, David L. (A)   Odom, O.W.  (A)   Shenkenberg, David  (A)   Garcia, Josh  (A)
Affiliations:	(A): Section of Molecular Cell and Developmental Biology, University of Texas at Austin
Web Site:	http://www.biosci.utexas.edu/mcdb/

Nearly 90% of the ~900 known group II introns are from chloroplast genes, yet no self-splicing group II intron from chloroplasts has been reported. Here we describe a large (2.6-kb) group II intron from the psbA gene (psbA1) of a psychrophilic Chlamydomonas that self-splices accurately in vitro. This intron, which also encodes an ORF with putative reverse transcriptase, maturase, and endonuclease domains, is related to group IIB2 introns from the cyanobacteria Calothrix and Nostoc; however, it also has distinctive features. In vitro self-splicing of psbA1 occurred via a lariat, and required Mg²⁺ (>12 mM) and monovalent salt. Self-splicing was improved by deleting most of the ORF, and by using pre-RNAs directly from transcription reactions, suggestive of a role for folding during transcription. Self-splicing of psbA1 pre-RNAs showed temperature optima of ~44 degrees C, but with a broad shoulder on the low side of the peak; splicing was nearly absent at 50 degrees C, indicative of thermolability. Splicing of wild-type psbA1 also occurred in E. coli, but not when the ORF was disrupted by mutations, providing genetic evidence that it has maturase activity. This work also provides the first description of a ribozyme from a psychrophilic organism.