Main content area

Reciprocal domain evolution within a tansactivator in a restricted sequence space

Juarez, K., Flores, H., Davila, S., Olvera, L., Gonzalez, V., Morett, E.
Proceedings of the National Academy of Sciences of the United States of America 2000 v.97 no.7 pp. 3314-3318
transcription factors, transcriptional activation, recombinant DNA, mutagenesis, Ensifer meliloti, beta-galactosidase, transcription (genetics), Bradyrhizobium japonicum, reporter genes, oxygen, DNA-binding domains, promoter regions
Although the concept of domain merging and shuffling as a major force in protein evolution is well established, it has been difficult to demonstrate how domains coadapt. Here we show evidence of coevolution of the Sinorhizobium meliloti NifA (SmNifA) domains. We found that, because of the lack of a conserved glycine in its DNA-binding domain, this transactivator protein interacts weekly with enhancers. This defect, however, was compensated by evolving highly efficient activation domain that, contrasting to Bradyrhizobium japonicum NifA (BjNifA), can activate in trans. To explore paths that lead to this enhanced activity, we mutagenized BiNifA. After three cycles of mutagenesis and selection, a highly active derivative was obtained. Strikingly, all mutations changed to amino acids already present in SmNifA. Our artificial process thus recreated the natural evolution followed by this protein and suggests that NifA is trapped in a restricted sequence space with very limited solutions for higher activity by point mutation.