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Mikhail Gelfand RECOMB-BE. UCSD, La Jolla, 15-III- 200 9

From computer to the wet lab and back Comparative genomics in the discovery of a family of bacterial transporters with a new mode of action. Mikhail Gelfand RECOMB-BE. UCSD, La Jolla, 15-III- 200 9. Transporters. Two main classes ATP-dependent TM-protein (permease) ATPase

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Mikhail Gelfand RECOMB-BE. UCSD, La Jolla, 15-III- 200 9

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  1. From computer to the wet lab and backComparative genomics in the discovery of a family of bacterial transporters with a new mode of action Mikhail Gelfand RECOMB-BE. UCSD, La Jolla, 15-III-2009

  2. Transporters • Two main classes • ATP-dependent • TM-protein (permease) • ATPase • Substrate-binding protein • Secondary (symporters, antiporters) • Difficult to study in experiment (compared to enzymes) • Relatively easy to identify • Similarity to known transporters • Prediction of transmembrane segments • Difficult to predict specificity H+

  3. It is difficult to predict specificity by sequence analysis (nickel-oligopeptide family,substrate-binding NikA)

  4. PnuC family of cofactor transporters

  5. Riboflavin biosynthesis pathway

  6. 5’ UTR regionsof riboflavin genes

  7. RFN-element Capitals: invariant (absolutely conserved) positions. Lower case letters: strongly conserved positions. Dashes and stars: obligatory and facultative base pairs Degenerate positions: R = A or G; Y = C or U; K = G or U; B= not A; V = not U. N: any nucleotide. X: any nucleotide or deletion

  8. RFN: the mechanism of regulation • Transcription attenuation • Translation attenuation

  9. YpaA: riboflavin transporter • 5 predicted TM segments => a transporter • Upstream RFN element => co-regulation with riboflavin genes => transport of riboflavin / precursor • S. pyogenes, E. faecalis, Listeria spp.: ypaA, no riboflavin pathway => transport of riboflavin Prediction:YpaA is riboflavin transporter(Gelfand et al., 1999) Verification: • by genetic analysis (Kreneva et al., 2000) • directly (Burgess et al., 2006) => RibU • ypaA is regulated by riboflavin(Lee et al., 2001) • … via attenuation of transcription(Winkler et al., 2003)

  10. Biotin transporter BioY • Identification: • co-localization • co-regulation • phylogenetic profiling • Additional components • ATPase(?) bioM • Permease(?) bioN

  11. = thiN (confirmed) Transport of HET Transport of HMP (Gram-positive bacteria) (Gram-negative bacteria) Thiamin biosynthesis

  12. yuaJ(=thiT): thiamine transporter • 6 predicted TM-segments • Regulated by THI riboswitches • Streptococci: ThiT, no thiamine pathway

  13. ykoFEDC: ATP-dependent HMP transporter • Regulated by THI riboswitches • Newer occurs in genomes lacking thiamine pathway • Always co-occurs with thiD and thiE • Sometimes occurs without thiC

  14. Cobalt andNickel • Co-localization • Ni transporterswith genes for Ni-dependent enzymes • Co transporters with cobalamine biosynthesis genes • Co-regulation • Ni transportersby transcription factorNikR • Co transportersby В12 riboswitich

  15. Structure of the loci genes B12 riboswitch NikR binding site

  16. Five families of transporters

  17. New ATP-dependent transporters + CbiN CbiM Ni2+ Co2+ NikM + NikN + NikL, NikK + NikL

  18. Dmitry Rodionov  Thomas Eitinger

  19. Test 1: predicted specificity is correct Co Co Ni Ni Ni Co

  20. Structure: too many components

  21. Biotin transporter BioY • ATPase BioM ~ CbiO = NikO • Permease BioN ~ CbioQ = NikQ

  22. Test 2: MN components aresuffucient(ATPase and permease are dispensable) cbiMNQO cbiMNQ cbiMN cbiM control

  23. Test 3: BioY is sufficienteven if the genome had BioMNY;BioMNY has better kynetics

  24. Tip of the iceberg?

  25. Validations FolT: folate (like BioY) RibU: riboflavin ThiT: thiamin

  26. Universal energizing component + specific components

  27. Transporters • Identification of candidates by analysis of transmembrane segments and similarity • Assignment to pathway by co-localization and co-regulation (in many genomes) • Prediction of specificity by analysis of phylogenetic patterns: • End product if present in genomes lacking this pathway (substituting the biosynthetic pathway for an essential compound) • Input metabolite if absent in genomes without the pathway (catabolic, also precursors in biosynthetic pathways) • Entry point in the middle if substituting an upper or side part of the pathway in some genomes

  28. Dmitry Rodionov • Alexei Vitreschak (RNA regulation) • Andrei Mironov (software, discussion) • Thomas Eitinger’s lab • Anrei Osterman’s lab • European Science Foundation • HHMI, RFBR, RAS, INTAS

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