Comparative genomic analysis of fungal TPP-riboswitches.

Riboswitches are conserved RNA structures located in non-coding regions of mRNA and able to bind small molecules (e.g. metabolites) changing conformation upon binding. This feature enables them to function as regulators of gene expression. The thiamin pyrophosphate (TPP) riboswitch is the only type of riboswitches found not only in bacteria, but also in eukaryotes - in plants, green algae, protists, and fungi. Two main mechanisms of fungal TPP riboswitch action, involving alternative splicing, have been established so far. Here, we report a large-scale bioinformatic study of riboswitch structural features, action mechanisms, and distribution along the fungal taxonomy groups. For each putatively regulated gene, we reconstruct the riboswitch structure, identify other components of the regulation machinery, and establish mechanisms of riboswitch-mediated regulation. In addition to three genes known to be regulated by TPP riboswitches, thiazole synthase THI4, hydroxymethilpyrimidine-syntase NMT1, and putative transporter NCU01977, we identify two new genes, a putative thiamin transporter THI9 and a transporter of unknown specificity. While the riboswitch sequence and structure remain highly conserved in all species and genes, the mode of riboswitch-mediated regulation varies between regulated genes. The riboswitch usage varies strongly between fungal taxa, with the largest number of riboswitch-regulated genes found in Pezizomycotina and no riboswitch-mediated regulation established in Saccaromycotina.

Comparative genomics and evolution of regulons of the LacI-family transcription factors.

DNA-binding transcription factors (TFs) are essential components of transcriptional regulatory networks in bacteria. LacI-family TFs (LacI-TFs) are broadly distributed among certain lineages of bacteria. The majority of characterized LacI-TFs sense sugar effectors and regulate carbohydrate utilization genes. The comparative genomics approaches enable in silico identification of TF-binding sites and regulon reconstruction. To study the function and evolution of LacI-TFs, we performed genomics-based reconstruction and comparative analysis of their regulons. For over 1300 LacI-TFs from over 270 bacterial genomes, we predicted their cognate DNA-binding motifs and identified target genes. Using the genome context and metabolic subsystem analyses of reconstructed regulons, we tentatively assigned functional roles and predicted candidate effectors for 78 and 67% of the analyzed LacI-TFs, respectively. Nearly 90% of the studied LacI-TFs are local regulators of sugar utilization pathways, whereas the remaining 125 global regulators control large and diverse sets of metabolic genes. The global LacI-TFs include the previously known regulators CcpA in Firmicutes, FruR in Enterobacteria, and PurR in Gammaproteobacteria, as well as the three novel regulators-GluR, GapR, and PckR-that are predicted to control the central carbohydrate metabolism in three lineages of Alphaproteobacteria. Phylogenetic analysis of regulators combined with the reconstructed regulons provides a model of evolutionary diversification of the LacI protein family. The obtained genomic collection of in silico reconstructed LacI-TF regulons in bacteria is available in the RegPrecise database (http://regprecise.lbl.gov). It provides a framework for future structural and functional classification of the LacI protein family and identification of molecular determinants of the DNA and ligand specificity. The inferred regulons can be also used for functional gene annotation and reconstruction of sugar catabolic networks in diverse bacterial lineages.