Characterization of a Group II Intron and its Intron-Encoded Protein
Abstract
Group II introns are a class of mobile genetic elements found in the genomes of bacteria, archaea and in the organellar genomes of some eukaryotes. They are comprised of a large structural RNA with ribozyme activity and a multi-functional intron-encoded protein. The IEP encodes four domains: reverse-transcriptase, maturase, DNA-binding and endonuclease domains. The RNA and IEP interact to form a functional ribonucleoprotien that facilitates the two activities of group II introns, splicing and mobility. The interaction between group IIA introns and their IEPs has been well characterized; however, many of the details regarding the IIC intron-IEP interaction are poorly understood. This dissertation examines the interaction between the B.h.I1 group II intron and its intron-encoded protein.
The B.h.I1 intron is a class IIC intron encoding an IEP that lacks the endonuclease domain. The intron is found in the organism Bacillus halodurans and exhibits clear evidence of retroelement behaviour, inserting downstream of intrinsic transcriptional terminators. In order to better characterize the intron-IEP interaction, it was determined that significant improvement to the IEP purification was required. Through a combination of optimization of expression and FPLC the yield and purity of the IEP was improved significantly. With purified components in sufficient quantity, the hypothesis that IIA and IIC IEPs recognize different high-affinity binding sites was examined using an in vitro mobility assay. Ultimately it was determined that IIC IEPs recognize a structurally similar but functionally different high-affinity binding site than is recognized by IIA-encoded IEPs. During binding site experiments, it was determined that the B.h.I1 intron uses the same IEP binding-site for splicing and mobility, reconciling a previously observed inconsistency.
A kinetic characterization found that the B.h.I1-IEP interaction occurs with lower affinity compared to the IIA intron and that the B.h.I1 intron-IEP interaction relies more heavily on secondary contacts made outside the high-affinity binding site. Finally, data obtained from cross-linking experiments implicate the IEP in recognition of the 3’ exon, a previously undiscovered function of the IEP in intron mobility. Though subtle, the differences observed for the IIC intron-IEP interaction appear to facilitate the specific requirements of its genomic niche.
Description
Keywords
Genetics, Biochemistry
Citation
Semper, C. (2016). Characterization of a Group II Intron and its Intron-Encoded Protein (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/25267