Tails of Three Knotty Switches: How PreQ1 Riboswitch Structures Control Protein Translation

Abstract

Riboswitches are structured RNA molecules that regulate genes by sensing cellular levels of metabolites such as preQ1 (7‐aminomethyl‐7‐deazaguanine). This economical platform is found predominantly in the 5′‐leader sequences of bacterial mRNAs, attracting attention as a potential antibiotic target. Members of the preQ1 riboswitch family provide exemplary insights into translational regulation because their high‐resolution structures have been determined, providing a framework to interpret complementary single‐molecule, computational and biochemical analyses. Although class I and II preQ1 riboswitches possess divergent pseudoknot architectures, both appear to function by burying the Shine‐Dalgarno sequence (SDS) within a core aptamer domain upon preQ1 binding. By contrast, the class III preQ1 riboswitch binds its ligand in an aptamer distant from the SDS. PreQ1 binding increases the population of riboswitches that transiently sequester the SDS, representing an alternative regulatory paradigm. Progress on preQ1 riboswitches is described, along with expectations for interfacing a riboswitch with the ribosome for translation initiation.

Key Concepts

  • Riboswitches are structured non‐protein‐coding RNAs that bind small molecules, tRNA or ions to control gene expression without the need for proteins.
  • Riboswitches bind protein enzyme co‐factors suggesting that they are remnants of an ancient RNA world.
  • Three classes of riboswitches bind the metabolite prequeuosine1 (preQ1), a pyrrolopyrimidine made exclusively in bacteria as a precursor to the hypermodified base queuosine, which is used widely in the biosphere to confer translational fidelity.
  • PreQ1 riboswitches adopt pseudoknot folds in which ligand binding stabilises coaxial helical stacking.
  • High‐resolution structures of class I, II and III preQ1 riboswitches reveal their overall three‐dimensional folds, along with chemical details that explain their high‐affinity ligand binding.
  • Structural observations suggest that riboswitches co‐opt traditional RNA tertiary interactions, such as major‐groove base‐triples and A‐minor motifs, for specialised biological functions.
  • Class I and II preQ1 riboswitches integrate Shine‐Dalgarno sequences (SDS) into their aptamer cores, producing gene off states in response to preQ1 binding.
  • Class III preQ1 riboswitches fold with spatially disparate aptamer and expression platform sequences that do not commingle in response to ligand binding.
  • Single‐molecule fluorescence resonance energy transfer (smFRET) provides insight into ligand‐dependent conformational changes of preQ1 riboswitches that control gene expression.
  • Modelling suggests that the class II preQ1 riboswitch must unfold only its anti‐SDS⋅SDS helix to interact with the 16S ribosomal RNA.

Keywords: preQ1 riboswitch; pseudoknot; gene regulation; ribosome; crystal structure; ligand recognition; Shine‐Dalgarno sequence; dynamics

Figure 1. Diagrams of riboswitch‐mediated gene regulation and interacting ligands. (a) Representative transcriptional and translation control by bacterial riboswitches. When ligand levels are low in the cell, signals in the 5′‐UTR of the mRNA favour transcription (i.e., an anti‐terminator stem‐loop is present) or translation [i.e., the Shine‐Dalgarno sequence (SDS) is accessible] of the downstream gene. When ligand levels rise, the aptamer binds the cognate effector shifting the population to a fold that alters expression platform accessibility in a manner that attenuates transcription or translation. (b) Chemical structures, names or abbreviations of molecules and ions that interact with known riboswitches – excluding tRNA. In some instances, an effector is recognised by multiple, distinct riboswitches [e.g., preQ1 is recognised by class I, II and III riboswitches (McCown et al., )], whereas others bind specific unique ligands including: ions, second messengers, nucleobases or derivatives thereof, enzyme co‐factors, or amino acids. Reviewed or described in Breaker, ; Nelson et al., ; McCown et al., ; Furukawa et al., ; Kellenberger et al., ; Kim et al., ; Nelson et al., .
Figure 2. Biosynthesis of queuosine (Q) and schematic diagrams of known preQ1 riboswitch classes. (a) Q synthesis occurs exclusively in bacteria and starts from GTP via the pyrrolopyrimidine intermediate preQ1; each arrow represents a separate enzymatic step. PreQ1 is the last free intermediate before insertion into the specific tRNAs shown, followed by further in situ modifications that generate Q. For a review see McCarty and Bandarian, . (b) The H‐type pseudoknot of the representative Thermoanaerobacter tengcongensis (Tte) preQ1‐I riboswitch. The pseudoknot comprises a five‐base‐pair stem (S1), a three‐nucleotide loop (L1), a three‐base‐pair S2, a four‐nucleotide L2 and a nine‐nucleotide L3 that joins the 3′‐most strands of each stem. Here and elsewhere a green oval represents preQ1; SDS nucleotides are highlighted (yellow). All pseudoknot diagrams are based on crystal structures in (e–g). (c) The HLout‐type pseudoknot of the representative Lactobacillus rhamnosus (Lra) preQ1‐II riboswitch. The pseudoknot begins with an eight‐base‐pair S1, followed by a five‐nucleotide L1, a six‐base‐pair S2, a single‐nucleotide L2 and an extended L3 of 19 nucleotides harbouring a stem‐loop. PreQ1‐II riboswitches typically exhibit a 5′‐stem‐loop outside the pseudoknot that is dispensable for preQ1 binding (Meyer et al., ; Kang et al., ) but was included in the Lra structure (grey). (d) The extended HLout‐type pseudoknot of the representative Faecalibacterium prausnitzii (Fpr) preQ1‐III riboswitch. The pseudoknot comprises a six‐base‐pair S1, a four‐nucleotide L1, a six‐base‐pair S2, a single‐nucleotide L2 and an extended L3 harbouring two stem‐loops comprising 26 and 29 nucleotides. The longer stem is capped by a loop labelled ‘anti‐SDS’ (aSDS) complementary to the 3′‐terminus of the riboswitch, including a portion of the SDS. (e) Ribbon diagram of the Tte preQ1‐I riboswitch crystal structure (PDB entry 3q50 from Jenkins et al., ). Here and elsewhere, the tertiary structure is coloured as depicted at left; preQ1 is depicted as a surface model. Ribose rings and base planes of the SDS are filled yellow. (f) Ribbon diagram of the Lra preQ1‐II riboswitch crystal structure (PDB entry 4jf2 from Liberman et al., ). (g) Ribbon diagram of the Fpr preQ1‐III riboswitch crystal structure (PDB entry 4rzd from Liberman et al., ).
Figure 3. Close‐up views of ligand‐binding pockets from preQ1 riboswitch family members that control translation. (a) The Tte preQ1‐I riboswitch (PDB entry 3q50 from Jenkins et al., ) shows canonical pairing between preQ1 and C15. (b) The Lra preQ1‐II riboswitch (PDB entry 4jf2 from Liberman et al., ) achieves ligand specificity via C30, which hydrogen bonds to the guanine‐like face of preQ1 using a reverse Watson–Crick pair, and U41, which recognises the ‘minor‐groove’ edge of preQ1 via alternating hydrogen‐bond donor and acceptor interactions. The SDS resides in the pocket floor and is accentuated by yellow, filled bases. (c) The Spn preQ1‐II riboswitch from the lowest‐energy NMR structure (PDB entry 4miy from Kang et al., ) exhibits a pocket homolgous to (b), except that a bifurcated hydrogen bond is observed between C8 and preQ1. (d) The Fpr preQ1‐III riboswitch (PDB entry 4rzd from Liberman et al., ) shows spatial similarity to the class II preQ1 riboswitch in (b), despite overall differences in the HLout pseudoknot and three‐dimensional fold (Figuref vs. Figureg). The all‐atom least‐squares superposition of (b) onto (d) (including preQ1 and 10 nucleotides lining the pocket – which considers the underlying U9⋅A86‐U15 major groove base triple) produced a root‐mean‐square deviation of 0.65 Å.
Figure 4. Schematic diagrams of preQ1 riboswitch conformations and dynamics from smFRET. (a) The Tte preQ1‐I riboswitch transitions from a mostly pre‐folded state with the 3′‐terminus docked (i.e. high‐FRET with few excursions into the mid‐FRET) into a compact, docked conformation upon preQ1 addition (Suddala et al., ). FRET pairs are denoted as green, red or blue circles; regions harbouring the SDS are highlighted (yellow); short lines indicate helical base pairs between long segments that represent the RNA backbone. (b) Conformation and dynamics of the Spn preQ1‐II riboswitch based on Soulière et al., (). PreQ1 favours formation of S2, which harbours the SDS and promotes formation of a high‐FRET state with few excursions to the undocked conformation. The extended stem‐loop in L3 remains dynamic regardless of preQ1. (c) Expression platform conformations of the Fpr preQ1‐III riboswitch. Percentages shown represent the population of molecules undergoing rapid docking and undocking of the 3′‐terminus that harbours the SDS under the indicated conditions. Although the riboswitch is depicted as folded in the left panel, SHAPE analysis suggests that S1 and S2 are flexible in the absence of preQ1. At any given time, only a small fraction of the population (∼14%) adopts the docked state. For details, see Liberman et al. ().
Figure 5. Overview of 70S ribosome complexes with bound tRNA and mRNA, and simple docking analysis of the Lra preQ1‐II riboswitch. (a) Crystal structure of 70S initiation‐like complex from Thermus thermophilus [PDB codes 3I9B and 3I9C (Jenner et al., )]. The small subunit (bottom) is shown in sky‐blue (RNA) and indigo (proteins); E‐ and P‐site tRNAs are shown. The aSDS⋅SDS helix forms between the 5′‐end of the mRNA and the 3′‐end of the 16S rRNA. The start codon (AUG) is positioned to pair with the P‐site tRNA. (b) Overlay of 16S rRNAs, mRNAs and E‐site tRNAs based on least‐squares superposition of the respective P‐site tRNAs from the initiation‐like complex in (a) and an elongation‐like complex [PDB codes 3I8F and 3I8G (Jenner et al., )]. The diagram illustrates the variability in the position of the aSDS⋅SDS helix. (c) Superposition of the Lra preQ1‐II riboswitch 5′‐S2 strand (i.e., the aSDS sequence) upon the 16S rRNA from the initiation‐like complex in (a) gives the position of riboswitch nucleotides 1–69. (By contrast, pairing of the riboswitch 5′‐S2 aSDS with that of the elongation‐like complex was prohibited sterically.) Inclined A‐minor nucleotide A70 was assumed as a hinge point (orange dot) that was manually rotated out of the ligand‐binding pocket as expected for the preQ1‐free state. Positions 71–76 of the riboswitch (i.e. the SDS) were oriented by superposition upon the SDS mRNA co‐crystallised in the elongation‐like complex shown in (b), which shares the common sequence 5′‐AGGAG‐3′. (d) Surface model of the ribosome coloured as in (a), and showing the fit of the S2‐unzipped preQ1‐II riboswitch into the cavity formed by the ribosome ‘head’ and ‘platform’. The 5′‐structure of the riboswitch is accommodated with only minor clashes with the N‐terminus of protein S18.
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References

Ames TD and Breaker RR (2011) Bacterial aptamers that selectively bind glutamine. RNA Biology 8 (1): 82–89.

Aytenfisu AH, Liberman JA, Wedekind JE and Mathews DH (2015) Molecular mechanism for PreQ1‐II riboswitch function revealed by molecular dynamics. RNA DOI: 10.1261/rna.051367.115.

Basile B and McCloskey J (1982) Queuine, a modified base incorporated posttranscriptionally into eukaryotic transfer RNA: wide distribution in nature. Science 216 (4541): 55–56.

Bienz M and Kubli E (1981) Wild‐type tRNATyrG reads the TMV RNA stop codon, but Q base‐modified tRNATyrQ does not. Nature 294: 188–190.

Breaker RR (2011) Prospects for riboswitch discovery and analysis. Molecular Cell 43 (6): 867–879.

Breaker RR (2012) Riboswitches and the RNA world. Cold Spring Harbor Perspectives in Biology 4 (2): a003566.

Crick F (1970) Central dogma of molecular biology. Nature 227 (5258): 561–563.

Crick FH (1958) On protein synthesis. Symposia of the Society for Experimental Biology 12: 138.

Furukawa K, Ramesh A, Zhou Z, et al. (2015) Bacterial riboswitches cooperatively bind Ni2+ or Co2+ ions and control expression of heavy metal transporters. Molecular Cell 57 (6): 1088–1098.

Grundy FJ and Henkin TM (1993) tRNA as a positive regulator of transcription antitermination in B. subtilis. Cell 74 (3): 475–482.

Harada F and Nishimura S (1972) Possible anticodon sequences of tRNAHis, tRNAAsn, and tRNAAsp from Escherichia coli. Universal presence of nucleoside Q in the first position of the anticodons of these transfer ribonucleic acids. Biochemistry 11 (2): 301–308.

Hiller DA and Strobel SA (2011) The chemical versatility of RNA. Philosophical Transactions of the Royal Society of London B: Biological Sciences 366 (1580): 2929–2935.

Jenkins JL, Krucinska J, McCarty RM, et al. (2011) Comparison of a preQ1 riboswitch aptamer in metabolite‐bound and free states with implications for gene regulation. Journal of Biological Chemistry 286 (28): 24626–24637.

Jenner LB, Demeshkina N, Yusupova G, et al. (2010) Structural aspects of messenger RNA reading frame maintenance by the ribosome. Nature Structural and Molecular Biology 17 (5): 555–560.

Kang M, Eichhorn CD and Feigon J (2014) Structural determinants for ligand capture by a class II preQ1 riboswitch. Proceedings of the National Academy of Sciences 111 (6): E663–E671.

Kang M, Peterson R and Feigon J (2009) Structural Insights into riboswitch control of the biosynthesis of queuosine, a modified nucleotide found in the anticodon of tRNA. Molecular Cell 33 (6): 784–790.

Kellenberger CA, Wilson SC, Hickey SF, et al. (2015) GEMM‐I riboswitches from Geobacter sense the bacterial second messenger cyclic AMP‐GMP. Proceedings of the National Academy of Sciences 112 (17): 5383–5388.

Kim PB, Nelson JW and Breaker RR (2015) An ancient riboswitch class in bacteria regulates purine biosynthesis and one‐carbon metabolism. Molecular Cell 57 (2): 317–328.

Klein DJ, Edwards TE and Ferré‐D'Amaré AR (2009) Cocrystal structure of a class I preQ1 riboswitch reveals a pseudoknot recognizing an essential hypermodified nucleobase. Nature Structural and Molecular Biology 16 (3): 343–344.

Korostelev A, Trakhanov S, Asahara H, et al. (2007) Interactions and dynamics of the Shine Dalgarno helix in the 70S ribosome. Proceedings of the National Academy of Sciences 104 (43): 16840–16843.

Liberman JA, Salim M, Krucinska J, et al. (2013) Structure of a class II preQ1 riboswitch reveals ligand recognition by a new fold. Nature Chemical Biology 9 (6): 353–355.

Liberman JA, Suddala KC, Aytenfisu AH, et al. (2015) Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome‐binding site regulated by fast dynamics. Proceedings of the National Academy of Sciences of the United States of America 112: E3484–E3494.

McCarty RM and Bandarian V (2008) Deciphering deazapurine biosynthesis: pathway for pyrrolopyrimidine nucleosides toyocamycin and sangivamycin. Chemistry & Biology 15 (8): 790–798.

McCown PJ, Liang JJ, Weinberg Z, et al. (2014) Structural, functional, and taxonomic diversity of three PreQ1 riboswitch classes. Chemistry & Biology 21 (7): 880–889.

Meier F, Suter B, Grosjean H, et al. (1985) Queuosine modification of the wobble base in tRNAHis influences' in vivo' decoding properties. The EMBO Journal 4 (3): 823.

Mellin J and Cossart P (2015) Unexpected versatility in bacterial riboswitches. Trends in Genetics 31 (3): 150–156.

Meyer MM, Roth A, Chervin SM, et al. (2008) Confirmation of a second natural preQ1 aptamer class in Streptococcaceae bacteria. RNA 14 (4): 685–695.

Mironov AS, Gusarov I, Rafikov R, et al. (2002) Sensing small molecules by nascent RNA: a mechanism to control transcription in bacteria. Cell 111 (5): 747–756.

Nahvi A, Sudarsan N, Ebert MS, et al. (2002) Genetic control by a metabolite binding mRNA. Chemistry & Biology 9 (9): 1043–1049.

Nelson JW, Sudarsan N, Furukawa K, et al. (2013) Riboswitches in eubacteria sense the second messenger c‐di‐AMP. Nature Chemical Biology 9 (12): 834–839.

Nelson JW, Sudarsan N, Phillips GE, et al. (2015) Control of bacterial exoelectrogenesis by c‐AMP‐GMP. Proceedings of the National Academy of Sciences 112 (17): 5389–5394.

Noguchi S, Nishimura Y, Hirota Y, et al. (1982) Isolation and characterization of an Escherichia coli mutant lacking tRNA‐guanine transglycosylase. Function and biosynthesis of queuosine in tRNA. Journal of Biological Chemistry 257 (11): 6544–6550.

Perrin DD (1965) Dissociation Constants of Organic Bases in Aqueous Solution (1972 Supplement). London: Butterworths.

Peselis A and Serganov A (2014) Structure and function of pseudoknots involved in gene expression control. Wiley Interdisciplinary Reviews: RNA 5 (6): 803–822.

Rakovich T, Boland C, Bernstein I, et al. (2011) Queuosine deficiency in eukaryotes compromises tyrosine production through increased tetrahydrobiopterin oxidation. Journal of Biological Chemistry 286 (22): 19354–19363.

Roth A, Winkler WC, Regulski EE, et al. (2007) A riboswitch selective for the queuosine precursor preQ1 contains an unusually small aptamer domain. Nature Structural and Molecular Biology 14 (4): 308–317.

Soulière MF, Altman RB, Schwarz V, et al. (2013) Tuning a riboswitch response through structural extension of a pseudoknot. Proceedings of the National Academy of Sciences 110 (35): E3256–E3264.

Spitale RC, Torelli AT, Krucinska J, et al. (2009) The structural basis for recognition of the PreQ0 metabolite by an unusually small riboswitch aptamer domain. Journal of Biological Chemistry 284 (17): 11012–11016.

Staple DW and Butcher SE (2005) Pseudoknots: RNA structures with diverse functions. PLoS Biology 3 (6): e213.

Suddala KC, Rinaldi AJ, Feng J, et al. (2013) Single transcriptional and translational preQ1 riboswitches adopt similar pre‐folded ensembles that follow distinct folding pathways into the same ligand‐bound structure. Nucleic Acids Research 41 (22): 10462–10475.

Suddala KC and Walter NG (2014) Riboswitch structure and dynamics by smFRET microscopy. Methods in Enzymology 549: 343–373.

Trausch JJ, Xu Z, Edwards AL, et al. (2014) Structural basis for diversity in the SAM clan of riboswitches. Proceedings of the National Academy of Sciences 111 (18): 6624–6629.

Urbonavičius J, Qian Q, Durand JM, et al. (2001) Improvement of reading frame maintenance is a common function for several tRNA modifications. The EMBO Journal 20 (17): 4863–4873.

van Batenburg FH, Gultyaev AP and Pleij CW (2001) PseudoBase: structural information on RNA pseudoknots. Nucleic Acids Research 29 (1): 194–195.

Wachter A (2014) Gene regulation by structured mRNA elements. Trends in Genetics 30 (5): 172–181.

Weinberg Z, Barrick JE, Yao Z, et al. (2007) Identification of 22 candidate structured RNAs in bacteria using the CMfinder comparative genomics pipeline. Nucleic Acids Research 35 (14): 4809–4819.

Weinberg Z, Regulski EE, Hammond MC, et al. (2008) The aptamer core of SAM‐IV riboswitches mimics the ligand‐binding site of SAM‐I riboswitches. RNA 14 (5): 822–828.

Winkler W, Nahvi A and Breaker RR (2002) Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419 (6910): 952–956.

Winkler WC and Breaker RR (2005) Regulation of bacterial gene expression by riboswitches. Annual Review of Microbiology 59: 487–517.

Yusupova G, Jenner L, Rees B, et al. (2006) Structural basis for messenger RNA movement on the ribosome. Nature 444 (7117): 391–394.

Further Reading

Eichhorn CD, Kang M and Feigon J (2014) Structure and function of preQ1 riboswitches. Biochimica et Biophysica Acta (BBA)‐Gene Regulatory Mechanisms 1839 (10): 939–950.

Garst AD, Edwards AL and Batey RT (2011) Riboswitches: structures and mechanisms. Cold Spring Harbor Perspectives in Biology 3 (6): a003533.

Li PTX, Vieregg J and Tinoco I (2008) How RNA unfolds and refolds. Annual Review of Biochemistry 77: 77–100.

Regulski EE and Breaker RR (2008) In‐line probing analysis of riboswitches. Methods in Molecular Biology 419: 53–67.

Rhodes G (2006) Crystallography Made Crystal Clear: A Guide for Users of Macromolecular Models, 3rd edn. Boston: Academic Press.

Robertson MP and Joyce GF (2012) The origins of the RNA world. Cold Spring Harbor Perspectives in Biology 4 (5): a003608.

Savinov A, Perez CF and Block SM (2014) Single‐molecule studies of riboswitch folding. Biochimica et Biophysica Acta (BBA)‐Gene Regulatory Mechanisms 1839 (10): 1030–1045.

Serganov A and Nudler E (2013) A decade of riboswitches. Cell 152 (1): 17–24.

Voorhees RM and Ramakrishnan V (2013) Structural basis of the translational elongation cycle. Annual Review of Biochemistry 82: 203–236.

Wilkinson KA, Merino EJ and Weeks KM (2006) Selective 2′‐hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single‐nucleotide resolution. Nature Protocols 1 (3): 1610–1616.

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Belashov, Ivan A, Dutta, Debapratim, Salim, Mohammad, and Wedekind, Joseph E(Oct 2015) Tails of Three Knotty Switches: How PreQ1 Riboswitch Structures Control Protein Translation. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021031]