Riboswitches: the oldest regulatory system?

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Riboswitches the oldest regulatory system?

• Mikhail Gelfand
• December 2004

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Riboflavin biosynthesis pathway
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5 UTR regions of riboflavin genes from various
bacteria
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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
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Attenuation of transcription
Antiterminator
Terminator
The RFN element
Antiterminator
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Attenuation of translation
Antisequestor
SD-sequestor
The RFN element
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RFN the mechanism of regulation

• Transcription attenuation

• Translation attenuation

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Distribution of RFN-elements
Genomes Number of analyzed genomes Number of genomes with RFN Number of the RFN elements
a-proteobacteria 8 4 4
ß-proteobacteria 7 4 4
?-proteobacteria 17 15 15
d- and e-proteobacteria 3 0 0
Bacillus/Clostridium 12 12 19
Actinomycetes 9 4 4
Cyanobacteria 5 0 0
Other eubacteria 7 5 6
Total 68 47 52
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Phylogenetic tree of RFN-elements
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YpaA riboflavin transporter in Gram-positive
bacteria

• 5 predicted transmembrane segments gt a
  transporter
• Upstream RFN element (likely co-regulation with
  riboflavin genes) gt transport of riboflaving or
  a precursor
• S. pyogenes, E. faecalis, Listeria sp. ypaA, no
  riboflavin pathway gt 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)

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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

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

• Transcription attenuation

• Bacillus/Clostridium group,
• Thermotoga,
• Fusobacterium,
• Chloroflexus

• Thermus/Deinococcus group,
• CFB group
• Proteobacteria,

• Translation attenuation

• Actinobacteria,
• Cyanobacteria,
• Archaea

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Distribution of THI-elements
Genomes Number of analyzed genomes Number of genomes with THI Number of the THI elements
a-proteobacteria 7 7 15
b-proteobacteria 6 6 12
g-proteobacteria 18 17 38
e- and d-proteobacteria 3 1 1
The Bacillus/Clostridium group 18 18 51
Actinomycetes 9 9 25
Cyanobacteria 5 5 5
Other eubacteria 14 11 11
Archaea (Thermoplasma) 17 3 6
Total 97 77 164
Mandal et al., 2003 THI in 3UTR (plants). THI
in untranslated intron (fungi)
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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

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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

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The PnuC family of transporters
The THI elements
The RFN elements
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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

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

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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

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Conserved RNA secondary structure of the
regulatory B12-element
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The predicted mechanism of the B12-mediated
regulation of cobalamin genes
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Distribution of B12-elements in bacterial genomes
B12-element regulates cobalamin biosynthetic
genes and transporters, cobalt transporters and a
number of other cobalamin-related genes.
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Metabolic reconstruction of cobalamin
biosynthesis new enzymes and transporters
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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 Ribonucleotide reductases
NrdJ (B12-dependent) NrdAB/NrdDG (B12-independent)



Methionine synthase Methionine synthase
MetH (B12-dependent) MetE (B12-independent)



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LYS-element lysine riboswitch
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Reconstruction of the lysine metabolism
predicted genes are boxed (pathway of acetylated
intermediates in B. subtilis)
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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
• gt acts as activator
• similar architecture of activating adenine
  riboswitch upstream of purine efflux pump ydhL
  (pbuE) in B. subtilis (Mandal and Breaker, 2004)

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S-box (SAM riboswitch)
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Reconstruction of the methionine metabolism
predicted genes are marked by (transport,
salvage cycle)
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A new family of amino acid transporters
S-box (rectangle frame)MetJ (circle
frame)LYS-element (circles)Tyr-T-box
(rectangles)
malate/lactate
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Regulation of reverse pathway Met-Cys in
Clostridium acetobutylicum
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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

MetJ, MetR in proteobacteria
ZOO
Lact.
Strep.
Bac.
Clostr.
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Riboswitches in the Sargasso sea metagenome

• 125 THI-elements
• 38 LYS-elements
• 25 B12-elements
• 9 RFN-elements
• 3 S-boxes

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Conserved structures of known riboswitches
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Characterized riboswitches (more are predicted)
RFN Riboflavin biosynthesis and transport FMN (flavin mononucleo-tide) Bacillus/Clostridium group, proteobacteria, actinobacteria, other bacteria
THI Biosynthesis and transport of thiamin and related compounds TPP (hiamin pyrophosphate) Bacillus/Clostridium group, proteobacteria, actinobacteria, cyanobacteria, other bacteria, archea (thermoplasmas), plants, fungi
B12 Biosynthesis of cobalamine, transport of cobalt, cobalamin-dependent enzymes Coenzyme B12 (adenosyl-cobalamin) Bacillus/Clostridium group, proteobacteria, actinobacteria, cyanobacteria, spirochaetes, other bacteria
S-box Metabolism of methionine and cystein SAM (S-adenosyl- methionine) Bacillus/Clostridium group and some other bacteria
LYS Lysine metabolism lysine Bacillus/Clostridium group, enterobacteria, other bacteria
G-box Metabolism of purines purines Bacillus/Clostridium group and some other bacteria
glmS Synthesis of glucosamine-6-phosphate glucosamine-6-phosphate Bacillus/Clostridium group
gcvT Catabolism of glycine glycine Bacillus/Clostridium group
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Mechanisms
gcvT ribozyme, cleaves its mRNA (the Breaker
group)
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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

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• 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