Bacterial Two-Hybrid Assay of Interactions between MatK and Chloroplast Proteins RNC1 and WTF1
Presentation Type
Poster
Full Name of Faculty Mentor
Michelle Barthet, Biology
Major
Biology
Presentation Abstract
Cancer can be caused by many different mutations in cells. Some of these mutations are the result of incorrect intron excision by splicing enzymes. Introns are extra nucleotide sequences that must be removed from precursor RNA to get the correct template for protein translation. Intron excision is important in the nucleus of animal and plant cells, as well as cellular organelles like plant chloroplasts which have their own gene expression machinery. Introns can be removed by three mechanisms: 1) the nuclear spliceosome, 2) self-excision, or 3) maturase enzymes. MatK is the only maturase encoded in the chloroplast of plants. Maturases are prokaryotic enzymes that bind and excise a single intron. MatK interacts with seven different introns instead of the single intron of prokaryotic maturases. Further, nuclear-encoded proteins like RNC1 and WTF1 excise the same introns as MatK suggesting the possible formation of a splicing complex. These evolutionary divergencies in number of intron targets and possible association of multiple proteins for facilitating intron excision are reminiscent of an early stage in nuclear spliceosome evolution. Bacterial two-hybrid assays will be used to assess protein interactions among MatK, and the two nuclear-encoded proteins WTF1 and RNC1, to characterize this potential chloroplast splicing complex. RT-PCR was used to amplify WTF1 and RNC1 coding regions followed by Gateway cloning into two-hybrid expression vectors and transformation into E. coli with interaction assayed by catalysis of a color substrate only expressed when the two target proteins interact.
Start Date
13-4-2023 12:00 PM
End Date
13-4-2023 2:00 PM
Disciplines
Biology
Recommended Citation
Hollins, Acacia and Presnell, Kirsten, "Bacterial Two-Hybrid Assay of Interactions between MatK and Chloroplast Proteins RNC1 and WTF1" (2023). Undergraduate Research Competition. 104.
https://digitalcommons.coastal.edu/ugrc/2023/fullconference/104
Bacterial Two-Hybrid Assay of Interactions between MatK and Chloroplast Proteins RNC1 and WTF1
Cancer can be caused by many different mutations in cells. Some of these mutations are the result of incorrect intron excision by splicing enzymes. Introns are extra nucleotide sequences that must be removed from precursor RNA to get the correct template for protein translation. Intron excision is important in the nucleus of animal and plant cells, as well as cellular organelles like plant chloroplasts which have their own gene expression machinery. Introns can be removed by three mechanisms: 1) the nuclear spliceosome, 2) self-excision, or 3) maturase enzymes. MatK is the only maturase encoded in the chloroplast of plants. Maturases are prokaryotic enzymes that bind and excise a single intron. MatK interacts with seven different introns instead of the single intron of prokaryotic maturases. Further, nuclear-encoded proteins like RNC1 and WTF1 excise the same introns as MatK suggesting the possible formation of a splicing complex. These evolutionary divergencies in number of intron targets and possible association of multiple proteins for facilitating intron excision are reminiscent of an early stage in nuclear spliceosome evolution. Bacterial two-hybrid assays will be used to assess protein interactions among MatK, and the two nuclear-encoded proteins WTF1 and RNC1, to characterize this potential chloroplast splicing complex. RT-PCR was used to amplify WTF1 and RNC1 coding regions followed by Gateway cloning into two-hybrid expression vectors and transformation into E. coli with interaction assayed by catalysis of a color substrate only expressed when the two target proteins interact.