structure and function of riboswitches

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A.Sri Devan mfrlab.org Lab meeting presentation 12/5/2010

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A review presentation on the structure and function of riboswitches.

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Page 1: Structure and function of riboswitches

A. Sri Devanmfrlab.org

Lab meeting presentation12/5/2010

Page 2: Structure and function of riboswitches

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

Page 3: Structure and function of riboswitches

Henkin T.M (2008) Genes & Development 22: 3383-3390

Page 4: Structure and function of riboswitches

Barrick & Breaker (2007) Genome Biology 8: R239

Page 5: Structure and function of riboswitches

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

Page 6: Structure and function of riboswitches

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)

Page 7: Structure and function of riboswitches

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

Page 8: Structure and function of riboswitches

(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

Page 9: Structure and function of riboswitches

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

Page 10: Structure and function of riboswitches

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

Page 11: Structure and function of riboswitches

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

Page 12: Structure and function of riboswitches

Sudarsan et al (2006) Science 314: 300-304

Page 13: Structure and function of riboswitches

Tertiary structures of riboswitches & RNA-ligand

interactions

Page 14: Structure and function of riboswitches

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

Page 15: Structure and function of riboswitches

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

Page 16: Structure and function of riboswitches

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

Page 17: Structure and function of riboswitches

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

Page 18: Structure and function of riboswitches

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

Page 19: Structure and function of riboswitches

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

Page 20: Structure and function of riboswitches

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

Page 21: Structure and function of riboswitches

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

Page 22: Structure and function of riboswitches

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

Page 23: Structure and function of riboswitches

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

Page 24: Structure and function of riboswitches

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

Page 25: Structure and function of riboswitches

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

Page 26: Structure and function of riboswitches

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

Page 27: Structure and function of riboswitches

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

Page 28: Structure and function of riboswitches

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