Recombinant Clematis ligusticifolia Maturase K (matK), partial

Introduction to Recombinant Clematis ligusticifolia Maturase K (matK), partial

Maturase K (matK) is a plastid-encoded group II intron maturase found in land plants . It plays a crucial role in the splicing of group II introns within plant organelles . The gene encoding matK is typically located within the intron of the transfer RNA (tRNA) gene trnk . The "recombinant" portion of the name signifies that the protein has been produced using recombinant DNA technology, involving the insertion of the matK gene into a host organism for expression and production. The "partial" descriptor indicates that the produced protein represents only a fragment of the full-length MatK protein. Clematis ligusticifolia is a vine-like plant, also known as Western clematis, and is the source of the matK gene in this instance .

Function and Importance of Maturase K

Maturase K is essential for the proper splicing of group II introns . Intron splicing is a vital process in gene expression, where non-coding sequences (introns) are removed from precursor RNA molecules, allowing the coding sequences (exons) to be joined to form mature RNA . MatK assists in the self-excision of introns in precursor RNAs .

Characteristics of MatK

MatK differs from other prokaryotic maturases because of the number of targets it binds . Compared to other prokaryotic-like maturases, MatK has lost two of the three main functional domains, specifically the C-terminal DNA endonuclease domain . It retains four of the seven reverse transcriptase (RT) domain sequence motifs . The RT0 and RT sequence motifs 1–4 contribute to RNA binding and target specificity .

Recombinant Production and Study

Recombinant MatK proteins are produced to study their activity and function in vitro . For example, a study used a recombinant form of MatK with a 6X His-tag, which allowed the protein to be isolated and purified for in vitro experiments examining its maturase activity .

Experimental Methods for Studying MatK Activity

To test MatK activity, scientists often use in vitro transcription to create precursor RNA substrates containing group II introns. These RNAs are then incubated with recombinant MatK protein under specific conditions, and the resulting products are analyzed to determine the extent of intron splicing .

Conditions for MatK activity assay:

• Heat denaturing of precursor RNA to unfold any previous tertiary structure

• Low magnesium concentration to prevent self-excision of targeted group IIA introns

• Incubation at 26°C for up to 60 minutes

Methods for Analyzing Splicing Activity:

• Quantitative PCR (qPCR): This technique is used to measure the levels of spliced product, un-spliced substrate, and total RNA at different time points .

• Reverse Transcription PCR (RT-PCR): RT-PCR is performed to confirm the presence of spliced products using intron-spanning primers .

• Controls: Controls, such as MatK protein alone and mock-induced E. coli protein, are used to ensure that the observed splicing activity is specifically due to MatK and not to contaminants .

Impact on Splicing

Studies have shown that MatK can significantly increase spliced product formation for certain RNAs, such as rps12-2, but not for others, such as rpl2 . This indicates that MatK exhibits some level of target specificity, even though it has lost certain functional domains found in other maturases .

Clematis ligusticifolia

Clematis ligusticifolia is a plant species used for medicinal purposes by Native Americans . Infusions from the plant have been used as a wash for skin eruptions and as a lotion for backaches . It can be found in riparian and run-on areas and is very drought tolerant .