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Riboswitches: the oldest regulatory system?

Riboswitches: the oldest regulatory system?. Mikhail Gelfand December 2004. Riboflavin biosynthesis pathway. 5 ’ UTR regions of riboflavin genes from various bacteria. Conserved secondary structure of the RFN-element. Capitals: invariant (absolutely conserved) positions.

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Riboswitches: the oldest regulatory system?

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  1. Riboswitches: the oldest regulatory system? Mikhail Gelfand December 2004

  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 gcvT: ribozyme, cleaves its mRNA (the Breaker group)

  41. 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

  42. 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

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