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Functional diversification of ROK-family transcriptional regulators of sugar catabolism in the Thermotogae phylum
Marat D. Kazanov
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1
Xiaoqing Li
1
Mikhail S. Gelfand
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Andrei L. Osterman
1
Dmitry A. Rodionov
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1
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A.A.Kharkevich Institute for Information Transmission Problems, Russian Academy of Sciences
,
Moscow 127994, Russia
1
Sanford-Burnham Medical Research Institute
,
La Jolla, CA 92037, USA
Large and functionally heterogeneous families of transcription factors have complex evolutionary histories. What shapes specificities toward effectors and DNA sites in paralogous regulators is a fundamental question in biology. Bacteria from the deep-branching lineage Thermotogae possess multiple paralogs of the repressor, open reading frame, kinase (ROK) family regulators that are characterized by carbohydrate-sensing domains shared with sugar kinases. We applied an integrated genomic approach to study functions and specificities of regulators from this family. A comparative analysis of 11 Thermotogae genomes revealed novel mechanisms of transcriptional regulation of the sugar utilization networks, DNA-binding motifs and specific functions. Reconstructed regulons for seven groups of ROK regulators were validated by DNA-binding assays using purified recombinant proteins from the model bacterium Thermotoga maritima. All tested regulators demonstrated specific binding to their predicted cognate DNA sites, and this binding was inhibited by specific effectors, mono- or disaccharides from their respective sugar catabolic pathways. By comparing ligand-binding domains of regulators with structurally characterized kinases from the ROK family, we elucidated signature amino acid residues determining sugar-ligand regulator specificity. Observed correlations between signature residues and the sugar-ligand specificities provide the framework for structure functional classification of the entire ROK family.
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DNA-binding transcription factors (TFs) in bacteria are
classified in at least 50 protein families based on sequence
similarity and domain composition (15). Prokaryotic TFs
are usually composed of two domains: (i) a DNA-binding
domain that provides the basic function in the recognition
of specific DNA sequences and (ii) an effector-sensing
domain that modulates the TF activity by monitoring
cellular signals and binding specific ligands. The
distribution of TFs by families varies among bacterial lineages,
mainly due to massive lineage-specific expansions of
specific TF families and frequent horizontal gene
transfer of individual TFs (6). Gene duplication followed
by functional diversification of the duplicated genes is a
major driver of evolution. Evolution of diverse
DNA-binding and ligand-binding activities in a given
TF family is a widely observed phenomenon. However,
our understanding of the evolutionary mechanisms
driving the observed diversity in large and functionally
heterogeneous families of TFs is very limited.
Several families of transcriptional regulators in bacteria
possess effector-sensing domains that are homologous to
ligand-binding domains of non-regulatory proteins. For
instance, the sugar-binding domains in regulators from
the LacI and DeoR families share the fold with
periplasmic binding proteins (PBPs) of sugar uptake
ABC transporters (7) and enzymes from the sugar
isomerase family (8), respectively. Structural similarity between
the effector-sensing domains of these regulators and the
ligand-binding domains of enzymes and transporters
suggests that the respective protein families are
evolutionarily related. Phylogenetic analysis of PBPs and LacI
family regulators revealed that the acquisition of the
DNA-binding domain occurred in the last common
ancestor of bacteria, and that both functional groups
have since undergone extensive gene duplication with
parallel evolution of ligand specificity (7). Whether similar
evolutionary scenarios could explain the origin of
functional divergence of ligand specificities in other TF
families remains an open question.
To understand the mechanisms of diversification of
ligand specificity, we selected the ROK (repressor, open
reading frame, kinase) protein family that includes two
functionally diverse groups of proteins: (i) catalytically
active sugar kinases and (ii) sugar-responsive
transcriptional repressors that possess an N-terminal
DNA-binding fused to a C-terminal sugar-binding
domain from the ROK family (9). The ROK protein
family is characterized by the PF00480 domain that
belongs to the Actin-ATPase clan in the Pfam database
(10). The broad distribution of this family in bacterial
genomes is illustrated by 9600 proteins in Pfam (on
August 2012). Among these, nearly 1700 proteins (18%)
are potential regulators that possess an N-terminal
DNA-binding domain. Some taxonomic groups of
bacteria, such as the deep-branching phylum Thermotogae
analyzed in this work, demonstrate lineage-specific
expansion of putative regulators from the ROK family.
However, the specificity and functional role of most
ROK regulators remain unknown. The only regulators
in this family that hav (...truncated)