Novel insights from hybrid LacI/GalR proteins: Family-wide functional attributes and biologically significant variation in transcription repression

Sarah Meinhardt, Michael W. Manley, Nicole A. Becker, Jacob A. Hessman, L. James Maher, Liskin Swint-Kruse

Research output: Contribution to journalArticle

40 Scopus citations

Abstract

LacI/GalR transcription regulators have extensive, non-conserved interfaces between their regulatory domains and the 18 amino acids that serve as 'linkers' to their DNA-binding domains. These non-conserved interfaces might contribute to functional differences between paralogs. Previously, two chimeras created by domain recombination displayed novel functional properties. Here, we present a synthetic protein family, which was created by joining the LacI DNA-binding domain/linker to seven additional regulatory domains. Despite 'mismatched' interfaces, chimeras maintained allosteric response to their cognate effectors. Therefore, allostery in many LacI/GalR proteins does not require interfaces with precisely matched interactions. Nevertheless, the chimeric interfaces were not silent to mutagenesis, and preliminary comparisons suggest that the chimeras provide an ideal context for systematically exploring functional contributions of non-conserved positions. DNA looping experiments revealed higher order (dimer-dimer) oligomerization in several chimeras, which might be possible for the natural paralogs. Finally, the biological significance of repression differences was determined by measuring bacterial growth rates on lactose minimal media. Unexpectedly, moderate and strong repressors showed an apparent induction phase, even though inducers were not provided; therefore, an unknown mechanism might contribute to regulation of the lac operon. Nevertheless, altered growth correlated with altered repression, which indicates that observed functional modifications are significant.

Original languageEnglish (US)
Pages (from-to)11139-11154
Number of pages16
JournalNucleic acids research
Volume40
Issue number21
DOIs
StatePublished - Nov 2012

ASJC Scopus subject areas

  • Genetics

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