In vivo evolution of a catalytic RNA couples trans-splicing to translation.
How does a non-coding RNA evolve in cells? To address this question experimentally we evolved a trans-splicing variant of the group I intron ribozyme from Tetrahymena over 21 cycles of evolution in E.coli cells. Sequence variation was introduced during the evolution by mutagenic and recombinative PCR, and increasingly active ribozymes were selected by their repair of an mRNA mediating antibiotic resistance. The most efficient ribozyme contained four clustered mutations that were necessary and sufficient for maximum activity in cells. Surprisingly, these mutations did not increase the trans-splicing activity of the ribozyme. Instead, they appear to have recruited a cellular protein, the transcription termination factor Rho, and facilitated more efficient translation of the ribozyme's trans-splicing product. In addition, these mutations affected the expression of several other, unrelated genes. These results suggest that during RNA evolution in cells, four mutations can be sufficient to evolve new protein interactions, and four mutations in an RNA molecule can generate a large effect on gene regulation in the cell.
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, United States of America.
Karen E
KE
Olson
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, United States of America.
Gregory F
GF
Dolan
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, United States of America.
Ulrich F
UF
Müller
D001483:Base Sequence; D002701:Chloramphenicol / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D019143:Evolution, Molecular; D007438:Introns / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics*; D016337:RNA, Catalytic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013768:Tetrahymena / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D020040:Trans-Splicing / Q000235:genetics*; D014158:Transcription, Genetic
C116167:GIR1 ribozyme; D016337:RNA, Catalytic; D012333:RNA, Messenger; D002701:Chloramphenicol; D015500:Chloramphenicol O-Acetyltransferase