A BACTERIAL GROUP-II INTRON ENCODING REVERSE-TRANSCRIPTASE, MATURASE,DNA ENDONUCLEASE ACTIVITIES - BIOCHEMICAL DEMONSTRATION OF MATURASE ACTIVITY AND INSERTION OF NEW GENETIC INFORMATION WITHIN THE INTRON

The Lactococcus lactis group II intron Ll.ltrB is similar to mobile ye ast mtDNA group II introns, which encode reverse transcriptase, RNA ma turase, and DNA endonuclease activities for site-specific DNA insertio n. Here, we show that the Lactococcal intron can be expressed and spli ced efficiently in Escherichia coli. The intron-encoded protein LtrA h as reverse transcriptase and RNA maturase activities, with the latter activity shown both in vivo and in vitro, a first for any group II int ron-encoded protein. As for the yeast mtDNA introns, the DNA endonucle ase activity of the Lactococcal intron is associated with RNP particle s containing both the intron-encoded protein and the excised intron RN A. Also, the intron RNA cleaves the sense-strand of the recipient DNA by a reverse splicing reaction, whereas the intron-encoded protein cle aves the antisense strand. The Lactococcal intron endonuclease can be obtained in large quantities by coexpression of the LtrA protein with the intron RNA in E. coli or reconstituted in vitro by incubating the expressed LtrA protein with in vitro-synthesized intron RNA. Furthermo re, the specificity of the endonuclease and reverse splicing reactions can be changed predictably by modifying the RNA component. Expression in E. coli facilitates the use of group II introns for the targeting of specific foreign sequences to a desired site in DNA.