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

Riboflavin biosynthesis pathway

5’ UTR regions of riboflavin genes from various bacteria

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

Attenuation of transcription Terminator The RFN element Antiterminator

Attenuation of translation SD-sequestor The RFN element Antisequestor

RFN: the mechanism of regulation Transcription attenuation Translation attenuation

Distribution of RFN-elements Genomes Number of analyzed genomes Number of genomes with RFN Number of the RFN elements α-proteobacteria844 β-proteobacteria744 γ-proteobacteria1715 δ- and ε-proteobacteria300 Bacillus/Clostridium12 19 Actinomycetes944 Cyanobacteria500 Other eubacteria756 Total684752

Phylogenetic tree of RFN-elements

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 )

More predicted (riboflavin) transporters impX from Fusobacterium and Desulfitobacterium –no similarity with any known protein; no homologs in other complete genomes –9 predicted TMS –single RFN-regulated gene pnuX from 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

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

Alignment of THI-elements

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

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

Distribution of THI-elements Genomes Number of analyzed genomes Number of genomes with THI Number of the THI elements  -proteobacteria 7715  -proteobacteria 6612  -proteobacteria  - and  proteobacteria 311 The Bacillus/Clostridium group18 51 Actinomycetes9925 Cyanobacteria555 Other eubacteria1411 Archaea (Thermoplasma)1736 Total Mandal et al., 2003: THI in 3’UTR (plants). THI in untranslated intron (fungi)

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-dependent HMP transporters Found in some Proteobacteria and Firmicutes; Not found in genomes without the thiamin pathway; Always co-occur with thiD and thiE; In Pasteurellae, Brucella and some 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

Predicted THI-regulated genes: more transporters thiU from P. multocida and H. influenzae belongs to the possible thiMDE-thiU operon, has 12 predicted TMS; similar to proline permease; no orthologs in other genomes thiV from Methylobacillus and H. volcanii clustered 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 thiW from S. pneumoniae and E. faecalis forms an operon with thiamin genes, has 5 predicted TMS; no homologs in other complete genomes pnuT from the CFB group of bacteria forms operon with thiamin-related genes; has 6 TMS; similar to the nicotinamide mononucleotide transporter PnuC from E.coli; no orthologs in other genomes cytX from Neiserria and Chloroflexus has 12 TMS, similar to the cytosine permease CodB from E. coli, forms an operon with thiamin genes in Neiserria and Pyrococcus; homologs in other genomes are not regulated by THI-elements. thiT1 and thiT2 from three 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

The PnuC family of transporters The RFN elements The THI elements

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; Forms clusters with thiD and 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 thiD and thiE genes 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. ylmB from Bacilli belongs 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 maritima belongs 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 Staphylococcus is a single THI-elements-regulated gene; belongs to the short-chain dehydrogenase/reductase (SDR) superfamily

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

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

B12-box and 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

Conserved RNA secondary structure of the regulatory B12-element

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

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

Metabolic reconstruction of cobalamin biosynthesis: new enzymes and transporters

If a bacterial genome contains B12-dependent and B12- independent isoenzymes, the genes encoding the B12- independent isoenzymes are regulated by B12-elements Ribonucleotide reductases NrdJ (B 12 -dependent (B 12 -dependent) NrdAB/NrdDG (B 12 -independent) +– –+ ++ Methionine synthase MetH (B 12 -dependent) MetE (B 12 -independent) +– –+ ++

LYS-element: lysine riboswitch

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

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)

S-box (SAM riboswitch)

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

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

Regulation of reverse pathway Met-Cys in Clostridium acetobutylicum

Three methionine regulatory systems in Gram-positive bacteria: loss of S-box regulons 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. ZOO MetJ, MetR in proteobacteria

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

Conserved structures of known riboswitches

Characterized riboswitches (more are predicted) RFNRiboflavin biosynthesis and transport FMN (flavin mononucleo-tide) Bacillus/Clostridium group, proteobacteria, actinobacteria, other bacteria THIBiosynthesis and transport of thiamin and related compounds TPP (hiamin pyrophosphate) Bacillus/Clostridium group, proteobacteria, actinobacteria, cyanobacteria, other bact., archaea (thermoplasmas), plants, fungi B12Biosynthesis of cobalamine, transport of cobalt, cobalamin-dependent enzymes Coenzyme B12 (adenosyl- cobalamin) Bacillus/Clostridium group, proteobacteria, actinobacteria, cyanobacteria, spirochaetes, other bacteria S-boxMetabolism of methionine and cystein SAM (S-adenosyl- methionine) Bacillus/Clostridium group and some other bacteria LYSLysine metabolismlysineBacillus/Clostridium group, enterobacteria, other bacteria G-boxMetabolism of purines purinesBacillus/Clostridium group and some other bacteria glmSSynthesis of glucosamine-6- phosphate glucosamine-6- phosphate Bacillus/Clostridium group gcvTCatabolism of glycineglycineBacillus/Clostridium group

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)

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

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

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