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Local gene regulation details a recognition code within the LacI transcriptional factor family.

ABSTRACT: The specific binding of regulatory proteins to DNA sequences exhibits no clear patterns of association between amino acids (AAs) and nucleotides (NTs). This complexity of protein-DNA interactions raises the question of whether a simple set of wide-coverage recognition rules can ever be identified. Here, we analyzed this issue using the extensive LacI family of transcriptional factors (TFs). We searched for recognition patterns by introducing a new approach to phylogenetic footprinting, based on the pervasive presence of local regulation in prokaryotic transcriptional networks. We identified a set of specificity correlations--determined by two AAs of the TFs and two NTs in the binding sites--that is conserved throughout a dominant subgroup within the family regardless of the evolutionary distance, and that act as a relatively consistent recognition code. The proposed rules are confirmed with data of previous experimental studies and by events of convergent evolution in the phylogenetic tree. The presence of a code emphasizes the stable structural context of the LacI family, while defining a precise blueprint to reprogram TF specificity with many practical applications.

SUBMITTER: Camas FM

PROVIDER: S-EPMC2978694 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Local gene regulation details a recognition code within the LacI transcriptional factor family.

Camas Francisco M FM Alm Eric J EJ Poyatos Juan F JF

PLoS computational biology 20101111 11

The specific binding of regulatory proteins to DNA sequences exhibits no clear patterns of association between amino acids (AAs) and nucleotides (NTs). This complexity of protein-DNA interactions raises the question of whether a simple set of wide-coverage recognition rules can ever be identified. Here, we analyzed this issue using the extensive LacI family of transcriptional factors (TFs). We searched for recognition patterns by introducing a new approach to phylogenetic footprinting, based on ...[more]

PMID: 21085639

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Project description:BACKGROUND: A key problem in the sequence-based reconstruction of regulatory networks in bacteria is the lack of specificity in operator predictions. The problem is especially prominent in the identification of transcription factor (TF) specific binding sites. More in particular, homologous TFs are abundant and, as they are structurally very similar, it proves difficult to distinguish the related operators by automated means. This also holds for the LacI-family, a family of TFs that is well-studied and has many members that fulfill crucial roles in the control of carbohydrate catabolism in bacteria including catabolite repression. To overcome the specificity problem, a comprehensive footprinting approach was formulated to identify TF-specific operator motifs and was applied to the LacI-family of TFs in the model gram positive organism, Lactobacillus plantarum WCFS1. The main premise behind the approach is that only orthologous sequences that share orthologous genomic context will share equivalent regulatory sites. RESULTS: When the approach was applied to the 12 LacI-family TFs of the model species, a specific operator motif was identified for each of them. With the TF-specific operator motifs, potential binding sites were found on the genome and putative minimal regulons could be defined. Moreover, specific inducers could in most cases be linked to the TFs through phylogeny, thereby unveiling the biological role of these regulons. The operator predictions indicated that the LacI-family TFs can be separated into two subfamilies with clearly distinct operator motifs. They also established that the operator related to the 'global' regulator CcpA is not inherently distinct from that of other LacI-family members, only more degenerate. Analysis of the chromosomal position of the identified putative binding sites confirmed that the LacI-family TFs are mostly auto-regulatory and relate mainly to carbohydrate uptake and catabolism. CONCLUSION: Our approach to identify specific operator motifs for different TF-family members is specific and in essence generic. The data infer that, although the specific operator motifs can be used to identify minimal regulons, experimental knowledge on TF activity especially is essential to determine complete regulons as well as to estimate the overlap between TF affinities.

Project description:Organisms regulate gene expression in response to the environment to coordinate metabolic reactions. Clostridium thermocellum expresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. One LacI regulator termed GlyR3 in C. thermocellum ATCC 27405 was previously identified as a repressor of neighboring genes with repression relieved by laminaribiose (a β-1,3 disaccharide). To better understand the three C. thermocellum LacI regulons, deletion mutants were constructed using the genetically tractable DSM1313 strain. DSM1313 lacI genes Clo1313_2023, Clo1313_0089, and Clo1313_0396 encode homologs of GlyR1, GlyR2, and GlyR3 from strain ATCC 27405, respectively. Growth on cellobiose or pretreated switchgrass was unaffected by any of the gene deletions under controlled-pH fermentations. Global gene expression patterns from time course analyses identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly upregulated (5- to 100-fold) in the absence of each LacI regulator, suggesting that these were repressed under wild-type conditions and that relatively few genes were controlled by each regulator under the conditions tested. Clo1313_2022, encoding lichenase enzyme LicB, was derepressed in a ΔglyR1 strain. Higher expression of Clo1313_1398, which encodes the Man5A mannanase, was observed in a ΔglyR2 strain, and α-mannobiose was identified as a probable inducer for GlyR2-regulated genes. For the ΔglyR3 strain, upregulation of the two genes adjacent to glyR3 in the celC-glyR3-licA operon was consistent with earlier studies. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters.IMPORTANCE Understanding C. thermocellum gene regulation is of importance for improved fundamental knowledge of this industrially relevant bacterium. Most LacI transcription factors regulate local genomic regions; however, a small number of those genes encode global regulatory proteins with extensive regulons. This study indicates that there are small specific C. thermocellum LacI regulons. The identification of LacI repressor activity for hemicellulase gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation in C. thermocellum.

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Local gene regulation details a recognition code within the LacI transcriptional factor family.

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Local gene regulation details a recognition code within the LacI transcriptional factor family.

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