A horizontally acquired group II intron in the chloroplast psbA gene of a psychrophilic Chlamydomonas: in vitro self-splicing and genetic evidence for maturase activity.
The majority of known group II introns are from chloroplast genomes, yet the first self-splicing group II intron from a chloroplast gene was reported only recently, from the psbA gene of the euglenoid, Euglena myxocylindracea. Herein, we describe a large (2.6-kb) group II intron from the psbA gene (psbA1) of a psychrophilic Chlamydomonas sp. from Antarctica that self-splices accurately in vitro. Remarkably, this intron, which also encodes an ORF with putative reverse transcriptase, maturase, and endonuclease domains, is in the same location, and is related to the E. myxocylindracea intron, as well as to group IIB2 introns from cyanobacteria. In vitro self-splicing of Chs.psbA1 occurred via a lariat, and required Mg(2+) (>12 mM) and NH(4)(+). 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 Chs.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 Chs.psbA1 also occurred in Escherichia coli, but not when the ORF was disrupted by mutations, providing genetic evidence that it has maturase activity. This work provides the first description of a ribozyme from a psychrophilic organism. It also appears to provide a second instance of interkingdom horizontal transfer of this group IIB2 intron (or a close relative) from cyanobacteria to chloroplasts.
Odom
Obed W
OW
Section of Molecular Cell and Developmental Biology and Institute for Cellular and Molecular Biology, School of Biological Sciences, University of Texas at Austin, 1 University Station A6700, Austin, TX 78712, USA.
Shenkenberg
David L
DL
Garcia
Joshua A
JA
Herrin
David L
DL
D000818:Animals; D000864:Antarctic Regions; D001483:Base Sequence; D015152:Blotting, Northern; D002696:Chlamydomonas / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D002736:Chloroplasts / Q000235:genetics*; D003001:Cloning, Molecular; D056890:Eukaryota / Q000235:genetics / Q000302:isolation & purification; D007438:Introns / Q000235:genetics*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010802:Phylogeny; D015800:Protozoan Proteins / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015800:Protozoan Proteins; D016053:RNA, Protozoan