Group II introns are self-splicing RNAs that also act as retroelements in bacteria, mitochondria, and chloroplasts. Group II introns were identified in Escherichia coli in 1994, but have not been characterized since, and, instead, other bacterial group II introns have been studied for splicing and mobility properties. Despite their apparent intractability, at least five distinct group II introns exist naturally in E. coli strains. To illuminate their function and learn how the introns have dispersed in their natural host, we have investigated their distribution in the ECOR reference collection. Two introns were cloned and sequenced to complete their partial sequences. Unexpectedly, southern blots showed all ECOR strains to contain fragments and/or full-length copies of group II introns, with some strains containing up to 15 intron copies. One intron, E.c.I4, has two natural homing sites in IS629 and IS911 elements, and the intron can be present in one, both, or neither homing site in a given strain. Nearly all strains that contain full-length introns also contain unfilled homing sites, suggesting either that mobility is highly inefficient or that most full-length copies are nonfunctional. The data indicate independent mobility of the introns, as well as mobility via the host DNA elements, and overall, the pattern of intron distribution resembles that of IS elements.

Telomeres in eukaryotic cells are generally synthesized and maintained by the ribonucleoprotein (RNP) telomerase. This enzyme is composed of at least two subunits, the telomerase reverse transcriptase (TERT) and the telomerase RNA. Human telomerase activity can be reconstituted in vitro by the expression of the telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. The hTERT and hTR subunits are independently expressed in vivo, and little is known about the mechanism of their assembly. To facilitate recombinant telomerase RNP formation and reconstitution, we engineered a construct, termed hTERT-hTR cis, in which the 3′ end of the hTERT coding sequence was extended by the addition of the sequence encoding hTR. Expression of the hTERT-hTR cis construct in vitro (in RRL) and in vivo (in the yeast Saccharomyces cerevisiae) produced hTERT-hTR transcripts of the predicted size. Active human telomerase was reconstituted by hTERT-hTR cis expression in both RRL and S. cerevisiae. Assembly of functional human telomerase by the bicistronic expression of the protein and RNA components may facilitate the overexpression and reconstitution of this enzyme in heterologous systems.

RNA transcripts corresponding to the 250-nt 3′ untranslated region of the R2 non-LTR retrotransposable element are recognized by the R2 reverse transcriptase and are sufficient to serve as templates in the target DNA-primed reverse transcription (TPRT) reaction. The R2 protein encoded by the Bombyx mori R2 can recognize this region from both the B. mori and Drosophila melanogaster R2 elements even though these regions show little nucleotide sequence identity. A model for the RNA secondary structure of the 3′ untranslated region of the D. melanogaster R2 retrotransposon was developed by sequence comparison of 10 species aided by free energy minimization. Chemical modification experiments are consistent with this prediction. A secondary structure model for the 3′ untranslated region of R2 RNA from the R2 element from B. mori was obtained by a combination of chemical modification data and free energy minimization. These two secondary structure models, found independently, share several common sites. This study shows the utility of combining free energy minimization, sequence comparison, and chemical modification to model an RNA secondary structure.