Recombinant Maturase K (matK)

Basic Research Questions

• What is Maturase K (matK) and what is its primary function in plant chloroplasts?

  Maturase K (MatK) is a plastid-encoded group II intron maturase found exclusively in land plants. It functions primarily as an enzyme that aids in the self-excision of group IIA introns in precursor RNAs within the chloroplast. MatK has evolutionary ties to the nuclear spliceosome and possesses a relatively high mutation rate at both nucleotide and amino acid levels, making it valuable for phylogenetic studies . Unlike typical prokaryotic maturases that target specific introns, MatK acts on multiple group IIA introns within the chloroplast genome, suggesting its evolution as a more general splicing factor. Research has demonstrated that MatK significantly increases spliced product formation for certain transcripts (e.g., rps12-2) by facilitating proper RNA folding into catalytically active structures needed for intron excision .

• How does MatK's structure differ from other prokaryotic maturases?

  MatK exhibits significant structural divergence from other prokaryotic maturases, particularly in its functional domains. While typical prokaryotic maturases contain three main functional domains (reverse transcriptase domain, domain X, and C-terminal DNA endonuclease domain), MatK has undergone substantial evolutionary modifications:

  • Contains only four of the seven reverse transcriptase (RT) domain sequence motifs

  • Has completely lost the DNA endonuclease domain

  • Retains the X domain, which is highly conserved and likely functions analogously to bacterial maturases in facilitating conformational RNA changes during splicing

  This domain reduction likely contributes to MatK's ability to bind multiple intron targets rather than being restricted to a single specific intron. The RT0 and RT sequence motifs 1-4 that MatK has retained are known to contribute to RNA binding and target specificity .

• What experimental evidence confirms MatK's role as a maturase in chloroplasts?

  Until recently, MatK's function was primarily inferred through indirect studies rather than direct experimental evidence. Key experimental approaches that have confirmed its maturase activity include:

  • In vitro activity assays using purified recombinant MatK protein with in vitro transcribed RNA substrates containing group IIA introns

  • Quantitative PCR measurements showing significantly increased spliced product formation (30-fold higher than self-splicing controls) when MatK was added to rps12 RNA

  • RT-PCR confirmation of spliced products using intron-spanning primers

  • Control experiments with mock-induced E. coli protein demonstrating that increased splicing resulted specifically from MatK activity

  These experiments directly demonstrated that MatK increases splicing efficiency of the group IIA intron in rps12-2, though interestingly, it had little effect on the splicing of the rpl2 intron, suggesting target specificity .

• Which chloroplast introns are targeted by MatK for splicing?

  MatK has been demonstrated to target multiple group IIA introns within the chloroplast genome. Based on research findings:

  • Significantly increases splicing efficiency of the rps12-2 intron

  • Shows little to no effect on the rpl2 intron, despite it containing a group IIA intron predicted to require MatK

  • Associated with the trnK intron, which has retained an open reading frame (ORF)

  The target specificity of MatK remains an active area of research, with evidence suggesting that it primarily acts on a subset of group IIA introns rather than all chloroplast introns .

• What protein interactions does MatK form during splicing?

  Recent research has revealed that MatK does not function alone but forms a complex with other proteins to facilitate splicing:

  • MatK physically interacts with a conserved, essential plastid-localized homolog of starch-branching enzymes (BEs), named MKIP1 (MatK Interacting Protein 1)

  • The complex formation may functionally compensate for the loss of MatK's N-terminal domain

  • This interaction likely helps MatK's initial binding to RNA and/or assists with intron folding into catalytically competent structures

  • The complex may provide MatK with a larger RNA binding surface, enabling it to dock to multiple introns via less specific contacts

  • The X domain of MatK remains free of MKIP1 interactions, allowing it to fit into the intron core and facilitate conformational RNA changes during splicing