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Nucleic Acids Res. 2012 Nov;40(21):11139-54. doi: 10.1093/nar/gks806. Epub 2012 Sep 10.

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

Author information

1
Department of Biochemistry and Molecular Biology, The University of Kansas Medical Center, Kansas City, KS 66160, USA.

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.

PMID:
22965134
PMCID:
PMC3505978
DOI:
10.1093/nar/gks806
[Indexed for MEDLINE]
Free PMC Article

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