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A. Sri Devanmfrlab.org
Lab meeting presentation12/5/2010
Riboswitches are cis-acting RNA elements which control gene expression by directly sensing the levels of specific small molecule metabolites.
Usually found in the 5’-untranslated region (5’-UTR) of mRNAs
Control a broad range of genes in bacterial species, including those involved in biosynthesis or transport of amino acids, cofactors, nucleotides & metal ions.
Edwards et al. (2007) Current Opinion in Structural Biology 17: 273-279Vitreschak et al. (2004) Trends in Genetics 20: 44-50
Nudler & Mironov (2004) Trends in Biochemical Sciences 29: 11-17
Henkin T.M (2008) Genes & Development 22: 3383-3390
Barrick & Breaker (2007) Genome Biology 8: R239
Firmicutes appear to the most extensive use of riboswitch classes where most aptamer classes occur multiple times per genome. For exp, B.subtilis carries at least 29 riboswitches (5 TPP, 1AdoCbl, 2 FMN, 1glycine, 11 SAM, 2 lysine, 1 GlcN6P, 4 guanine, 1 adenine & 1 preQ1) controlling approximately 73 genes.
γ-Proteobacteria employ a mixture of these riboswitch classes that is comparable to the diversity found in Firmicutes species.
Riboswitches also seem to occur only rarely in Chlamydia species, Cyanobacteria, and Spirochetes.
Barrick & Breaker (2007) Genome Biology 8: R239
Typically composed of 2 domains; aptamer domain & expression platform
Aptamer domain serves as a molecular sensor that selectively recognizes its target molecule
Binding of the target molecule is signaled to the expression platform that interfaces with the transcriptional & translational RNA machinery to regulate gene expression either through
- formation of a transcription terminator or - formation of a helical structure that sequesters the Shine
Dalgarno sequence (SD)
Pre-Queuosine
2’-deoxyguanosine
Winkler & Breaker (2005) Annu Rev Microbiol 59: 487-517
Roth et al. (2007) Nat Struct Mol Biol. 14: 308-317
Kim et al. (2007) Proc. Natl. Acad. Sci. 104: 16092-16097
(Left) In the absence of the effector, the aptamer domain is unoccupied, & the RNA is in a conformation that allows expression of downstream coding sequence either through formation of an anti terminator helix that allows transcription to continue (top) or through formation of a helical structure that liberates the SD sequence and allows translation to initiate (bottom). The reverse happens when the aptamer domain is in the occupied stage (right)
Henkin T.M (2008) Genes & Development 22: 3383-3390
The B. subtilis glmS (glucosamine synthase) riboswitch is conserved upstream of the glms gene
During conditions of excess GlcN6P (glucosamine-6-phosphate), the glmS 5’-UTR is stimulated to self-cleave at its 5’-end (indicated by the arrow)
Cleavage leads to glmS repression through an unknown mechanism
Winkler W.C (2005) Current Opinion in Chemical Biology 9: 594-602
Glycine riboswitch from V.cholerae was found to sense intracellular glycine through cooperative binding
In glycine absence, helix 2+3 is formed which inhibit ribosome access
During conditions of glycine excess, glycine bind to the first domain and increases the affinity for the second domain through cooperative interactions
Once both aptamers are occupied, alternative helix 1+2 forms allowing ribosome access and translation to initiate
Winkler W.C (2005) Current Opinion in Chemical Biology 9: 594-602
Two aptamer domains or even two complete switches can lie adjacent to each other, resulting in a more complex mechanism of gene expression regulation.
Examples include ; 1. Tandem glycine aptamers which bind glycine cooperatively (as previously explained). 2. Tandem arrangement of 2 different riboswitches (such as SAM & AdoCbl) that independently sense different metabolites, and funtions as a 2-input Boolean NOR logic gate wherein binding of either ligand causes repression. 3. Tandem arrangement of 2 identical riboswitches (such as TPP) which would enable a greater responsiveness to changes in metabolite concentration.
Sudarsan et al (2006) Science 314: 300-304
Sudarsan et al (2006) Science 314: 300-304
Tertiary structures of riboswitches & RNA-ligand
interactions
There are 2 variants of purine riboswitch; adenine & guanine ribowitch
The RNA folds into three helices, P1, P2 and P3 which are arranged as an inverted ‘h’
P1 stacks coaxially under P3, and P2 & P3 pack side-by-side
This overall arrangement is stabilized by the interaction of the terminal loops of P2 & P3
Edwards et al. (2007) Current Opinion in Structural Biology 17: 273-279Serganov et al. (2004) Chem Biol 11: 1729-1741
Purine riboswitches contain a ligand binding pocket situated between several layers of base triples (not shown) which constitute the 3-way junction
The purine ligand is primarily recognized by residue 74 of the riboswitch, a pyrimidine through Watson Crick pairing
C74: the riboswitch binds to guanineU74: the riboswitch binds to adenine
Edwards et al. (2007) Current Opinion in Structural Biology 17: 273-279
Winkler W.C (2005) Current Opinion in Chemical Biology 9: 594-602
TPP (thiamine pyrophosphate) is the biologically active form of thiamine
TPP riboswitch adopts a compact, inverted h architecture with 2 parallel sets of coaxially stacked helices (P1-P2-P3 & P4-P5) joined by a 3-way junction
Interaction between residues of L5 and P3 stabilizes this arrangement
Edwards et al. (2007) Current Opinion in Structural Biology 17: 273-279Serganov et al. (2006) Nature 441: 1167-1171
Thore et al. (2006) Science 312: 1208-1211
The only riboswitch that has been found in eukaryotes - it is found as part of an intron in fungi - it is found within 3’UTR of plants
The structures of TPP-riboswitches from bacterial and plant origin are highly similar
Can be used in different regulatory contexts - inhibition of translation & premature transcription termination in Gram + & Gram – bacteria - splicing control in fungi - postulated control of processing or stability of plant mRNAs
Sudarsan et al. (2003) RNA 9: 644-647
Miranda-Rios (2007) Structure 15: 259-265
Kubodera et al. (2003) FEBS Lett 555: 516-520
The 2 helical stacks of the thi-box riboswitch separately recognize the pyrimidine & pyrophosphate moities of TPP
The pyrimidine-like ring pairs with G40 and is stacked between G42 & A43.
The pyrophosphate sensor helix (the P4-P5 stack) recognizes the pyrophosphate moiety of TPP through interaction with solvated divalent cations (Mg2+)
Edwards et al. (2007) Current Opinion in Structural Biology 17: 273-279
The structure features a complex FMN bound junctional region stapled together by two peripheral domains, P2-P6 & P3-P5.
Each peripheral domain is formed by two interacting stem-loops, stabilized by 2 pairs of tertiary contacts involving loop-loop (L2-L6 & L3-L5) & loop helix (L6-P2 & L3-P5) interactions resulting in a butterfly like scaffold
Serganov A. (2009) Current Opinion in Structural Biology 19: 251-259
Serganov et al. (2009) Nature 458: 233-237
The ligand FMN orients its extremities toward different domains of the riboswitch
The ring structure is sandwiched between between purines (A48 & A85) and is involved in specific hydrogen bonding with conserved A99
The phosphate moiety forms the majority of hydrogen bonds with several conserved guanines
Edwards et al. (2007) Current Opinion in Structural Biology 17: 273-279
One of the largest known riboswitch classes
Features three helical and two helical bundles radiating from a compact 5-way helical junction
Stems P2 & P3 are aligned by kissing loop interactions between loops L2 & L3
Parallel stems are P2 & P4 are joined by a conserved loop (L4)-helix (P2) contact
Edwards et al. (2007) Current Opinion in Structural Biology 17: 273-279
Serganov et al. (2008) Nature 455: 1263-1267
Lysine is positioned in the middle layer of a tight pocket & is surrounded by evolutionary conserved nucleotides
The carboxylate & ammonium groups of the lysine ‘main-chain’ segment recognize purine bases & sugar phosphate backbone respectively
A notable feature of the lysine binding pocket is a K+ cation which binds a carbonyl oxygen of lysine & zippers up the binding pocket using several coordination bonds
Serganov A. (2009) Current Opinion in Structural Biology 19: 251-259
3 different variants exist - SAM-I - SAM-II - SAM-III (or SMK)
All 3 different variants of SAM riboswitches have distinct consensus sequence & secondary structure
Species from different bacteria lineages appear to rely on distinct classes of SAM-sensing riboswitches to control key sulfur metabolic pathways
Wang & Breaker (2008) Biochem. Cell Biol 86: 157-168
SAM-III riboswitch folds into an inverted Y-shaped molecule, centred on the SAM bound 3-way junction
The SAM-II riboswitch comprises a continous helix P1/P2b/P2a & 2 loops (L1 & L3) interacting with the grooves of P2a/P2b & P1
The SAM-1 riboswitch contains 2 helical stacks (P1-P4 and P2a-P3) which cross at an angle of ~70°. A pseudoknot atop P2 appears to stablize this fold
SAM-II SAM-I SAM-III
Serganov A. (2009) Current Opinion in Structural Biology 19: 251-259
Lu et al. (2008) Nature Structural & Molecular Biology 15: 1076-1083
Montage & Batey (2006) Nature 441: 1172-1175Gilbert et al. (2008) Nature Structural & Molecular Biology 15: 177-182
Edwards et al. (2007) Nature Structural & Molecular Biology 17: 273-279
Hydrogen bonding patterns of SAM recognition varies in all three riboswitches
The ligand adopts distinct conformations in different SAM-riboswitches. For instance, in SAM-I riboswitch, SAM appears in a compact form whereas in SAM-II, it is in extended form
Common features include - stacking of adenine moiety with RNA bases - electrostatic interactions of the positively charged sulfur moiety with O4 carbonyls of uracils.
Serganov A. (2009) Current Opinion in Structural Biology 19: 251-259
The shortest known riboswitch that controls biosynthesis of the modified nucleoside present in certain tRNAs
Folds into an H-type pseudoknot
The tertiary structure of this RNA comprises of two stems (S1 & S2) separated by three loops (L1, L2 & L3)
Loops L1 & L3 lie in the major and minor grooves of stems S2 & S1
Klein et al (2009) Nature Structural & Molecular Biology 16: 343-344
Intercalation of preQ1 at the interhelical interface (between G11 & the G5-C18 pair) allows efficient coaxial stacking of S1 & S2 stems
In addition pairing to C17, preQ1 also pairs with A30 & U6
Furthermore the aminomethyl group of preQ1 is recognized through specific H-bonding witha)G5b)phosphate oxygen of G11c)hydration water of Ca2+
Klein et al (2009) Nature Structural & Molecular Biology 16: 343-344
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