Mikhail Gelfand Research and Training Center “Bioinformatics”

1. Comparative genomic analysis of T-box regulation: identification of new structural classes and reconstruction of evolution Mikhail Gelfand Research and Training Center “Bioinformatics” Institute for Information Transmission Problems Moscow, Russia HHMI Conference, June 2008

2. T-boxes: the mechanism (Grundy & Henkin; Putzer & Grunberg-Manago)

3. Partial alignment of predicted T-boxes TGG: T-box Aminoacyl-tRNA synthetases Amino acid biosynthetic genes Amino acid transporters

4. … continued (in the 5’ direction) anti-anti (specifier) codon Aminoacyl-tRNA synthetases Amino acid biosynthetic genes Amino acid transporters

5. Why T-boxes? • May be easily identified • In most cases functional specificity may be reliably predicted by the analysis of the specifier codons (anti-anti-codons) • Sufficiently long to retain phylogenetic signal => T-boxes are a good model of regulatory evolution

6. 805 T-boxes in 96 bacteria • Firmicutes • aa-tRNA synthetases • enzymes • transporters • all amino acids excluding glutamate • Actinobacteria (regulation of translation – predicted) • branched chain (ileS) • aromatic (Atopobium minutum) • Delta-proteobacteria • branched chain (leu – enzymes) • Thermus/Deinococcus group (aa-tRNA synthases) • branched chain (ileS, valS) • glycine • Chloroflexi, Dictyoglomi • aromatic (trp – enzymes) • branched chain (ileS) • threonine

7. Double and partially double T-boxes • TRP: trp operon (Bacillales, C. beijerincki, D. hafniense) • TYR: pah (B. cereus) • THR: thrZ (Bacillales); hom (C. difficile) • ILE: ilv operon (B. cereus) • LEU: leuA (C. thermocellum) • ILE-LEU: ilvDBNCB-leuACDBA (Desulfotomaculum reducens) • TRP: trp operon (T. tengcongensis) • PHE: arpLA-pheA (D. reducens, S. wolfei) • PHE: trpXY2 (D. reducens) • PHE: yngI (D. reducens) • TYR: yheL (B. cereus) • SER: serCA (D. hafniense) • THR: thrZ(S. uberis) • THR: brnQ-braB1 (C. thermocellum) • HIS: hisXYZ (Lactobacillales) • ARG: yqiXYZ (C. difficile)

8. Predicted regulation of translation:ileS in many Actinobacteria • Instead of the terminator, the sequester hairpin (hides the translation initiation site) • Same mechanism regulates different processes – cf. riboswitches

9. A new type of translational T-boxes in Actinobacteria • Shorter specifier hairpin • Anti-anti-codon in the “head” loop, not a bulge loop • A majority of cases (all except Streptomyces spp.)

10. Same enzymes – different regulators (common part of the aromatic amino acids biosynthesis pathway) cf. E.coli: aroF,G,H: feedback inhibition by TRP, TYR, PHE; transcriptional regulation by TrpR, TyrR

11. Recent duplications and bursts: ARG-T-box in Clostridium difficile

12. … caused by loss of transcription factor AhrC

13. Duplications and changes in specificity: ASN/ASP/HIS T-boxes

14. Blow-up 1

15. Blow-up 2. Prediction Regulators lost in lineages with expanded HIS-T-box regulon??

16. … and validation • conserved motifs upstream of HIS biosynthesis genes • candidate transcription factor yerC co-localized with the his genes • present only in genomes with the motifs upstream of the his genes • genomes with neither YerC motif nor HIS-T-boxes: attenuators Bacillales(his operon) Clostridiales Thermoanaerobacteriales Halanaerobiales Bacillales

17. New histidine transporters • hisXYZ(The ATP-binding Cassette (ABC) Superfamily)Firmicutes • yuiF(Na+/H+ antiporter, NahC family)Bacillales, some Clostridiales(regulated by his-attenuator in Haemophilus inlfuenzae) • Cphy_3090(SSS sodium solute transporter superfamily)Clostridiales, Thermoanaerobacteriales, Halanaerobiales

18. The evolutionary history of the his genes regulation in the Firmicutes

19. More duplications: THR-T-box in C. difficileand B. cereus

20. Duplications and changes in specificity: branched-chain amino acids ATC CTC ATC

21. Blow-up transporter: ATC GTC dual regulation of common enzymes: ATC CTC

22. Summary / History

23. Otherresults • Bacteria (comparative genomics of regulation) • Reconstruction of metabolic pathways and their regulation • niacin • ethanolamine • Prediction of regulation • cysteine and methionine pathways in the Streptococcus spp. • radiation resistance in the Deinococcus spp. • Identification and experimental validation (collaborators) of a new class of transporters with shared ATP-dependent energizing modules • Identification of new microcins • Analysis of co-evolution of transcription factors and their binding motifs • Eukaryotes (alternative splicing) • Evolution of the exon-intron structure and alternative splicing in the Drosophila spp. and in mammals • estimates of the rate of intron and exon gain and loss • Proof of positive selection in minor-isoform alternative regions of human genes

24. Acknowledgements • Alexei Vitreschak • Ekaterina Ermakova • Alexei Kazakov • Marat Kazanov • Galina Kovaleva • Andrei Mironov • Ramil Nurtdinov • Mikhail Pyatnitsky • Alexandra Rakhmaninova • Dmitry Ravcheev • Valery Sorokin • Olga Tsoy • Anna Gerasimova (Ann-Arbor) • Olga Kalinina (Heidelberg) • Dmitry Rodionov (La Jolla) • Thomas Eitinger (Berlin) • Dmitry Malko (Moscow) • Andrey Osterman (La Jolla) • Vasily Ramensky (Moscow) • Konstantin Severinov (Moscow) • HHMI • RFBR • RAS (program “Molecular and Cellular Biology”)