Defining 5S rRNA structure space: point mutation data can be used to predict the phenotype of multichange variants.

A portion of the 5S ribosomal RNA (rRNA) structure space in the vicinity of the Vibrio proteolyticus 5S rRNA sequence is explored in detail with the intention of establishing principles that will allow a priori prediction of which sequences would be valid members of a particular RNA structure space. Four hundred and one sequence variants differing from the V. proteolyticus 5S rRNA wild-type sequence in 1-7 positions were characterized using an in vivo assay system. Most significantly, it was found that in general, the phenotypic effects of single changes were independent of the phenotypic effect of a second change. As a result, it was possible to use the new data in conjunction with results from prior studies of the same RNA to develop "truth tables" to predict which multiple change variants would be functional and which would be nonfunctional. The actual phenotype of 93.8% of the multichange variants studied was consistent with the predictions made using truth tables thereby providing for perhaps the first time an upper limit estimate of how frequent unexpected interactions are. It was also observed that single changes at positions involved in secondary structure were no more likely to be invalid than changes in other regions. In particular, internal changes in long standard stems were in fact almost always tolerated. Changes at positions that were hypervariable in the context of an alignment of related sequences were, as expected, usually found to be valid. However, the potential validity of changes that were idiosyncratic to a single lineage of related sequences when placed in the V. proteolyticus 5S rRNA context was unpredictable.

Rapid in vivo exploration of a 5S rRNA neutral network.

A partial knockout compensation method to screen 5S ribosomal RNA sequence variants in vivo is described. The system utilizes an Escherichia coli strain in which five of eight genomic 5S rRNA genes were deleted in conjunction with a plasmid which is compensatory when carrying a functionally active 5S rRNA. The partial knockout strain is transformed with a population of potentially compensatory plasmids each carrying a randomly generated 5S rRNA gene variant. a The ability to compensate the slow growth rate of the knockout strain is used in conjunction with sequencing to rapidly identify variant 5S rRNAs that are functional as well as those that likely are not. The assay is validated by showing that the growth rate of 15 variants separately expressed in the partial knockout strain can be accurately correlated with in vivo assessments of the potential validity of the same variants. A region of 5S rRNA was mutagenized with this approach and nine novel variants were recovered and characterized. Unlike a complete knockout system, the method allows recovery of both deleterious and functional variants.. The method can be used to study variants of any 5S rRNA in the E. coli context including those of E. coli.