Bacterial group II introns are large catalytic RNAs related to nuclear spliceosomal introns and eukaryotic retrotransposons. They self-splice to yield mature RNA, and integrate into DNA as retroelements. A fully active group II intron forms a ribonucleoprotein complex comprising the intron ribozyme and an intron-encoded protein, with multiple activities including reverse transcriptase. This activity is responsible for copying the intron RNA into the DNA target. Here we report cryo-EM structures of an endogenously spliced Lactococcus lactis group IIA intron in its ribonucleoprotein complex form at 3.8 Å resolution and in its protein-depleted form at 4.5 Å resolution, revealing functional coordination of the intron RNA with the protein. Remarkably, the protein structure reveals a close relationship of the reverse transcriptase catalytic domain to telomerase, whereas the active center for splicing resembles the spliceosomal Prp8 protein. These extraordinary similarities hint at intricate ancestral relationships and provide new insights into splicing and retromobility.

SummaryGroup II introns are mobile retroelements that invade their cognate intron-minus gene in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. Previous studies of the Lactococcus lactis intron Ll.LtrB indicated that in its native host, as in Escherichia coli , retrohoming occurs by the intron RNA reverse splicing into double-stranded DNA (dsDNA) through an endonucleasedependent pathway. However, in retrotransposition in L. lactis , the intron inserts predominantly into single-stranded DNA (ssDNA), in an endonucleaseindependent manner. This work describes the retrotransposition of the Ll.LtrB intron in E. coli , using a retrotransposition indicator gene previously employed in our L. lactis studies. Unlike in L. lactis , in E. coli , Ll.LtrB retrotransposed frequently into dsDNA, and the process was dependent on the endonuclease activity of the intron-encoded protein.Further, the endonuclease-dependent insertions preferentially occurred around the origin and terminus of chromosomal DNA replication. Insertions in E. coli can also occur through an endonucleaseindependent pathway, and, as in L. lactis , such events have a more random integration pattern. Together these findings show that Ll.LtrB can retrotranspose through at least two distinct mechanisms and that the host environment influences the choice of integration pathway. Additionally, growth conditions affect the insertion pattern. We propose a model in which DNA replication, compactness of the nucleoid and chromosomal localization influence target site preference.

Inteins are mobile genetic elements that self-splice at the protein level. Mycobacteria have inteins inserted into several important genes, including those corresponding to the iron-sulfur cluster assembly protein SufB. Curiously, the SufB inteins are found primarily in mycobacterial species that are potential human pathogens. Here we discovered an exceptional sensitivity of Mycobacterium tuberculosis SufB intein splicing to oxidative and nitrosative stresses when expressed in Escherichia coli. This effect results from predisposition of the intein's catalytic cysteine residues to oxidative and nitrosative modifications. Experiments with a fluorescent reporter system revealed that reactive oxygen species and reactive nitrogen species inhibit SufB extein ligation by forcing either precursor accumulation or N-terminal cleavage. We propose that splicing inhibition is an immediate, posttranslational regulatory response that can be either reversible, by inducing precursor accumulation, or irreversible, by inducing N-terminal cleavage, which may potentially channel mycobacteria into dormancy under extreme oxidative and nitrosative stresses.

Group II introns are mobile retroelements that invade their hosts. The Lactococcus lactis group II intron recruits cellular polymerases, nucleases, and DNA ligase to complete the retromobility process in Escherichia coli. Here we describe a genetic screen with a Tn5 transposon library to identify other E. coli functions involved in retromobility of the L. lactis LtrB intron. Thirteen disruptions that reproducibly resulted in increased or decreased retrohoming levels into the E. coli chromosome were isolated. These functions were classified as factors involved in RNA processing, DNA replication, energy metabolism, and global regulation. Here we characterize a novel mutant in the rne promoter region, which regulates RNase E expression. Retrohoming and retrotransposition levels are elevated in the rneTTn5 mutant. The stimulatory effect of the mutation on retromobility results from intron RNA accumulation in the RNase E mutant. These results suggest that RNase E, which is the central component of the RNA degradosome, could regulate retrohoming levels in response to cellular physiology.