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

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.

Applications and advances of metabolite biosensors for metabolic engineering

“…In one example, an isopentenyl diphosphate (IPP) biosensor was created by fusing the AraC DNA binding domain and the linker with IPP isomerase Idi (Chou and Keasling, 2013). These general approaches are useful to develop sensors for a variety of compounds, as demonstrated for triacetic acid lactone (Tang et al, 2013), D-arabinose (Tang et al, 2008), fructose, ribose (Meinhardt et al, 2012;Shis et al, 2014), etc.…”

Flexibility and Disorder in Gene Regulation: LacI/GalR and Hox Proteins

“…The flexibility of these regions is critical to strong transcription repression and allosteric regulation. B, the flexible interface between the LacI/GalR linkers and regulatory domains can facilitate allosteric response to multiple ligands (79). The LacI DNA-binding domain/linker can be fused to the regulatory domains of other homologs (shown as schematic dimers) to create functional chimeric repressors (red bars in graph) with intact allosteric response to small effector ligands (blue bars).…”

“…The LacI DNA-binding domain/linker can be fused to the regulatory domains of other homologs (shown as schematic dimers) to create functional chimeric repressors (red bars in graph) with intact allosteric response to small effector ligands (blue bars). Note that the "LLHP" chimera has the opposite allosteric response of LacI (79). DEL control indicates the activity of reporter enzyme in the absence of repressor.…”

“…In fact, fully functional hybrids were created by fusing the LacI DNA-binding domain/ linker to regulatory domains from other homologs. Each chimera has the DNA binding specificity of LacI, ligand binding of the parent regulatory domain, and allosteric response defined by the regulatory domain (79). Thus, the interface between the linker and regulatory domains is highly adaptable.…”

AlloRep: A Repository of Sequence, Structural and Mutagenesis Data for the LacI/GalR Transcription Regulators

“…The LacI/GalR paralogs have evolved specificities for different DNA sequences and allosteric effectors [20]. Although domain recombination shows that the allosteric mechanism may largely be the same, the magnitude and direction of allosteric response can be modulated [20,21]. In general, predicting the locations of allosteric positions has been challenging.…”