1. Institute for Information Transmission Problems
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. May be easily identified
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)
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. difficile and B. cereus

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

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

22. Summary / History

23. Other results 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”)