Date of Award

1-1-2009

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (xv, 256 pages)

Dissertation/Thesis Chair

Marlene Belfort

Committee Members

Keith Derbyshire, Joan Curcio, Janice Pata, Robert Osuna

Keywords

group II intron, host factors, retrotransposition, retrotransposon, RNA, Genetic engineering, Introns

Subject Categories

Molecular Biology

Abstract

Group II introns are mobile retroelements. They invade the cognate intron-minus gene in an efficient process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. Retrohoming occurs by the intron RNA reverse-splicing into double-stranded DNA (dsDNA) through an endonuclease-dependent pathway. However, in retrotransposition in Lactoccocus lactis, the intron inserts predominantly into single-stranded DNA (ssDNA), in an endonuclease-independent manner. Unlike in L. lactis, in Escherichia coli the Ll.LtrB intron retrotransposes frequently into dsDNA, and the process is dependent on the endonuclease activity of the intron-encoded protein. Further, the endonuclease-dependent integrations preferentially occurred around the origin and terminus of chromosomal DNA replication. These macrodomains migrate toward the poles of the cell, where the intron-encoded protein, LtrA, localizes. Integrations in E. coli can also occur through an endonuclease-independent pathway, and, as in L. lactis, such events have a more random integration pattern. Our findings show that Ll.LtrB can retrotranspose through at least two distinct mechanisms and that the host environment influences the choice of integration pathway. We investigated whether alteration of nucleoid condensation, chromosome partitioning and replication affect retrotransposition frequencies, as well as bipolar localization of the Ll.LtrB intron and LtrA in E. coli. Although there were some dramatic fluctuations in retrotransposition levels, bipolar localization of integration events was maintained. LtrA was consistently found in nucleoid-free regions, with its localization to the cellular poles being largely preserved in these hosts. Together, these results suggest that bipolar localization of group II intron retrotransposition results from the residence of the intron-encoded protein at the poles of the cell. The group II Ll.LtrB intron resides on the pRS01 conjugative plasmid of L. lactis, in the ltrB gene, which encodes a conjugative relaxase. Intron splicing is necessary to produce a functional relaxase, LtrB, which initiates conjugation by nicking the DNA at the origin of transfer (oriT). We show that pRS01 and relaxase stimulates retrotransposition. LtrF, a DNA-binding protein that, like LtrB, is an essential component of the relaxosome, also stimulated retrotransposition. These results indicate that the relaxosome enhances retrotransposition, providing a rationale for the residence of group II introns on a conjugative mobile element. Collectively, my studies show that the lifestyle of the group II intron is influenced by the host and by circumstances prevailing in the bacterial cell.

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