Riboswitches: the oldest regulatory system?

1. Riboswitches: the oldest regulatory system? Mikhail Gelfand Research and Training Center on Bioinformatics Institute for Information Transmission Problems Russian Academy of Sciences BITS Annual Meeting. Milan, March 2005

2. Riboflavin biosynthesis pathway

3. 5’ UTR regionsof riboflavin genes from various bacteria

4. Conserved secondary structure of the 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

5. Attenuation of transcription Antiterminator Terminator The RFN element Antiterminator

6. Attenuation of translation Antisequestor SD-sequestor The RFN element

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

8. Distribution of RFN-elements

9. Phylogenetic tree of RFN-elements

10. YpaA: riboflavin transporter in Gram-positive bacteria • 5 predicted transmembrane segments => a transporter • Upstream RFN element (likely co-regulation with riboflavin genes) => transport of riboflaving or a precursor • S. pyogenes, E. faecalis, Listeria sp.: ypaA, no riboflavin pathway => transport of riboflavin Prediction: YpaA is riboflavin transporter (Gelfand et al., 1999) Verification: • YpaA transports flavines (riboflavin, FMN, FAD) (by genetic analysis, Kreneva et al., 2000) • ypaA is regulated by riboflavin (by microarray expression study, Lee et al., 2001) • … via attenuation of transcription (and to some extent inhibition of translaition) (Winkler et al., 2003)

11. More predicted (riboflavin) transporters impXfromFusobacterium and Desulfitobacterium • no similarity with any known protein; no homologs in other complete genomes • 9 predicted TMS • single RFN-regulated gene pnuXfrom Actinomycetes(Corynebacterium, Streptomyces, Thermomonospora) • no orthologs in other genomes • 6 predicted TMS • either a single gene or a part of the riboflavin operon • regulated by RFN • similar to the nicotinamide mononucleotide transporter PnuC from E. coli

12. thi-boxand regulation of thiamine metabolism genes by pyrophosphate (Miranda-Rios et al., 2001)

13. Alignment of THI-elements

14. Conserved secondary structure of the THI-element Capitals: strongly conserved positions. Dashes and points: obligatory and facultative base pairs Degenerate positions: R = A or G; Y = C or U; K = G or U; M= A or C; N = any nucleotide

15. THI: the mechanism of regulation • Transcription attenuation • Bacillus/Clostridium group, • Thermotoga, • Fusobacterium, • Chloroflexus • Thermus/Deinococcus group, • CFB group • Proteobacteria, • Translation attenuation • Actinobacteria, • Cyanobacteria, • Archaea

16. Distribution of THI-elements Mandal et al., 2003: THI in 3’UTR (plants). THI in untranslated intron (fungi)

17. Predicted THI-regulated genes: transporters yuaJ: predicted thiamin transporter (possibly H+-dependent) • Found only in the Bacillus/Clostridium group; • Occurs in genomes without the thiamin pathway (Streptococci); • Has 6 predicted transmembrane segments (TMS); • Regulated by THI-elements in all cases with only one exception (E. faecalis); • In B. cereus, the thiamin uptake is coupled to proton movement (Arch Microbiol, 1977). thiX-thiY-thiZ and ykoF-ykoE-ykoD-ykoC: predicted ATP-dependentHMP transporters • Found in some Proteobacteria and Firmicutes; • Not found in genomes without the thiamin pathway; • Always co-occur with thiDandthiE; • In Pasteurellae, Brucellaandsome Gram-positive cocci,they are present without thiC; • Regulated by THI-elements in all cases with only one exception (T. maritima); • Putative substrate-binding protein ThiY is homologous to Thi12 from yeast, known to be involved in the biosynthesis of HMP

18. Predicted THI-regulated genes: more transporters • thiUfromP. multocidaandH. influenzaebelongs to the possible thiMDE-thiU operon, has 12 predicted TMS; similar to proline permease; no orthologs in other genomes • thiVfrom Methylobacillus and H. volcaniiclustered with thiamin genes or has THI-elements,has 13 predicted TMS , similar to the pantothenate symporter PanF from E.coli; no orthologs in other genomes • thiWfrom S. pneumoniaeandE. faecalis forms an operon with thiamin genes, has 5 predicted TMS; no homologs in other complete genomes • pnuTfrom the CFB group of bacteriaforms operon with thiamin-related genes; has 6 TMS;similar to the nicotinamide mononucleotide transporter PnuC from E.coli; no orthologs in other genomes • cytXfromNeiserria and Chloroflexushas 12 TMS, similar to the cytosine permease CodB from E. coli, forms an operon with thiamin genes in Neiserriaand Pyrococcus; homologs in other genomes arenot regulated by THI-elements. • thiT1 and thiT2fromthree different Thermoplasma(Archaea)are two paralogous genes; have 9 TMS; belong to the MFS family of transporters. This is the first example of THI-element-regulated genes in Archaea

19. The PnuC family of transporters The THI elements The RFN elements

20. Predicted THI-regulated genes: enzymes • thiN: non-orthologous displacement of thiE Separate gene in archaea or with thiD (in M. theroautotrophicum) Always present if ThiD is present and ThiE is absent • tenA: gene of unknown function somehow associated with thiD Found in most firmicutes, some proteobacteria and archaea; ThiD-TenA gene fusions in some eukaryotes; Formsclusters with thiDand other THI-elements-regulated genes in most bacteria; Single tenA gene is also regulated by THI-elements in some bacteria; Not found in genomes without the thiamin pathway; Always co-occurs with the thiDandthiEgenes • tenI: gene of unknown function, thiE paralog Found in some unrelated bacteria; Forms a separate branch in the phylogenetic tree for thiE; In most bacteria, located in clusters of THI-elements-regulated genes. • ylmBfrom Bacillibelongs to the ArgE/dapE/ACY1/CPG2/yscS family of metallopeptidases; regulated by the THI-elements in B. subtilis and B. halodurans, not regulated in B. cereus. • thi-4 from Thermotoga maritimabelongs to a family of putative thiamine biosynthetic enzymes from archaea and eukaryotes. Located in the one operon with thiC and thiD. • oarX from Methylobacillus and Staphylococcusis a single THI-elements-regulated gene; belongs to the short-chain dehydrogenase/reductase (SDR) superfamily

21. Metabolic reconstruction of the thiamin biosynthesis = thiN (confirmed) Transport of HET Transport of HMP (Gram-positive bacteria) (Gram-negative bacteria)

22. THI-elements in delta-proteobacteria: co-operative binding? • Tandem arrangement of THI-elements upstream of the main thiamine operon thiSGHFE1 in Desulfovibrio spp. • Tandem arrangement of glycine riboswitches in B. subtilis and V. cholerae (Mandal et al., 2004): • co-operative binding of the cofactor (glycine) • rapid activation/repression • same arrangement in all glycine riboswitches

23. B12-boxand regulation of cobalamin metabolism genes by pyrophosphate (Nou & Kadner, 2000; Ravnum & Andersson, 2001; Nahvi et al., 2002) • Long mRNA leader is essential for regulation of btuB by vitamin B12. • Involvement of highly conserved B12-box rAGYCMGgAgaCCkGCcd in regulation of the cobalamin biosynthetic genes (E. coli, S. typhimurium) • Post-transcriptional regulation: RBS-sequestering hairpin is essential for regulation of the btuB and cbiA • Ado-CBL is an effector molecule involved in the regulation of the cobalamin biosynthesis genes

24. Conserved RNA secondary structure of the regulatory B12-element

25. The predicted mechanism of the B12-mediated regulation of cobalamin genes

26. Distribution of B12-elements in bacterial genomes B12-elementregulates cobalamin biosynthetic genes and transporters, cobalt transporters and a number of other cobalamin-related genes.

27. Metabolic reconstruction of cobalamin biosynthesis: new enzymes and transporters

28. If a bacterial genome contains B12-dependent and B12-independent isoenzymes, the genes encoding the B12-independent isoenzymes are regulated by B12-elements

29. LYS-element: lysine riboswitch

30. Reconstruction of the lysine metabolism predicted genes are boxed (pathway of acetylated intermediates in B. subtilis)

31. Regulation of lysine catabolism: the first example of an activating riboswitch • LYS-elements upstream of pspFkamADEatoDA operon in Thermoanaerobacter tengcongensis; kamADElysE operon in Fusobacterium nucleatum • lysine catablism pathway • LYS element overlaps candidate terminator => acts as activator • similar architecture of activating adenine riboswitch upstream of purine efflux pump ydhL (pbuE) in B. subtilis (Mandal and Breaker, 2004)

32. S-box (SAM riboswitch)

33. Reconstruction of the methionine metabolism predicted genes are marked by *(transport, salvage cycle)

34. S-box (rectangle frame)MetJ (circle frame)LYS-element (circles)Tyr-T-box (rectangles) A new family of amino acid transporters malate/lactate

35. Regulation of reverse pathway Met-Cys in Clostridium acetobutylicum

36. Three methionine regulatory systems in Gram-positive bacteria: loss of S-box regulons MetJ, MetR in proteobacteria ZOO • S-boxes (riboswitch) • Bacillales • Clostridiales • the Zoo: • Petrotoga • actinobacteria (Streptomyces, Thermobifida) • Chlorobium, Chloroflexus, Cytophaga • Fusobacterium • Deinococcus • proteobacteria (Xanthomonas, Geobacter) • Met-T-boxes (Met-tRNA-dependent attenuator) • Lactobacillales • MET-boxes(transcription factor MtaR) • Streptococcales Lact. Strep. Bac. Clostr.

37. Riboswitches in the Sargasso sea metagenome • 125 THI-elements • 38 LYS-elements • 25 B12-elements • 9 RFN-elements • 3 S-boxes

38. Conserved structures of known riboswitches

39. Characterized riboswitches (more are predicted)

40. Mechanisms glmS: ribozyme, cleaves its mRNA (the Breaker group)gcvT: co-operative riboswitches(the Breaker group)THI in plants: required for splicing (Kubodera et al., 2003)

41. Structure of the purine riboswitch(Noeske et al. 2004)(see also Serganov et al., 2004)

42. Properties of riboswitches • Direct binding of ligands • Same structure – different mechanisms • Distribution in all taxonomic groups • diverse bacteria • archaea - thermoplasmas • eukaryotes – plants and fungi • Lineage-specific features… • … horizontal transfer, duplications, lineage-specific loss • Correlation of the mechanism and taxonomy: • attenuation of transcription (anti-anti-terminator) – Bacillus/Clostridium group • attenuation of translation (anti-anti-sequestor of translation initiation) – proteobacteria • attenuation of translation (direct sequestor of translation initiation) – actinobacteria • splicing – eukaryotes

43. Andrei Mironov • software genome analysis, conserved RNA patterns • Alexei Vitreschak • analysis of RNA structures • Dmitry Rodionov • metabolic reconstruction • Support: • Howard Hughes Medical Institute • INTAS • Russian Fund of Basic Research • Russian Academy of Sciences