Bacterial gene regulation: metal ion sensing by proteins or RNA

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TRENDS in Biotechnology

Vol.24 No.9

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Bacterial gene regulation: metal ion sensing by proteins or RNA Sabine Brantl AG Bakteriengenetik, Friedrich-Schiller-Universita¨t Jena, Philosophenweg 12, D-07743, Jena, Germany

Until recently, metal sensing in bacteria seemed to be accomplished exclusively by metalloregulatory proteins; however, a surprising new finding is that a metal ion itself can act as a riboswitch ligand to shut down gene expression. Interestingly, this ion is Mg2+, known to be required for a wide variety of cellular functions and for correct folding of RNAs. It remains to be discovered whether other ion-dependent riboswitches exist, which would open up a new dimension for regulatory RNAs. Introduction Several mechanisms of gene regulation in bacteria have been discovered to date, among them transcriptional control by DNA-binding proteins, post-transcriptional control exerted by cis- or trans-encoded regulatory RNAs [1,2] or through the modulation of RNA structure. The latter mechanism, initially termed transcription attenuation, was discovered 25 years ago in the E. coli tryptophan operon [3]. Since then, it has been observed and intensively studied in several systems, and a variety of attenuation mechanisms have been found [4]. Characteristic of all these cases is the ability of RNA to fold into two alternative structures: one promotes transcription termination, whereas the other enables read-through (anti-termination). For example, anti-termination is supported by uncharged tRNAs in the tRNA synthetase operons of Gram-positive bacteria, whereas tryptophan-bound TRAP (trp RNAbinding attenuation protein) promotes transcription termination in the B. subtilis trp operon [4], and small antisense RNAs mediate transcription attenuation of rep mRNAs in the Inc18 plasmids [1]. Modulation of RNA structures: riboswitches open up a new dimension In 2002, two studies reported the first experimental evidence for gene regulation through direct binding of small metabolites to structured 50 leader regions of mRNA termed ‘riboswitches’ [5,6]. One of these riboswitches supported a transcription attenuation mechanism in which alternative RNA folding is induced by binding of flavin mononucleotide (FMN) to the leader region of nascent riboflavin mRNA in B. subtilis [5]. The other so-called translational riboswitch represents a mechanism found mainly in Gram-negative bacteria and acts to inhibit translation of the E. coli thiamine mRNA through sequestration of the Shine-Dalgarno (SD) sequence [6]. Interestingly, traditional transcription attenuation and the newly Corresponding author: Brantl, S. ([email protected]). Available online 26 July 2006. www.sciencedirect.com

described ligand-dependent riboswitches both mediate regulation through alternative RNA secondary structures. In traditional attenuation, translation of a leader reading frame and tRNA charging are sensed, leading to premature transcription termination. By contrast, riboswitch binding of a small metabolite induces an alternative RNA fold that leads to premature transcription termination or – for translational riboswitches – sequestration of the SD sequence. Unbound RNAs fold into an anti-terminator structure, enabling transcription or translation to proceed. During the past four years, a remarkable variety of transcriptional and translational riboswitches were found in evolutionarily diverse bacterial species [7,8] (Table 1), where they regulate genes involved in numerous metabolic pathways. Among them, peculiarities are the B. subtilis adenine activator riboswitch, the glycine riboswitch with two aptamer domains and the glmS riboswitch that even functions as a ribozyme [9]. All these examples show that RNA has sufficient structural diversity to form highly specific binding pockets for a multitude of ligands. However, despite publication of the first crystal structures of the guanine riboswitch [8], the elucidation of the molecular mechanisms by which the different riboswitches work is still in its infancy. Metal sensing by proteins that act as transcriptional regulators Metals are required as cofactors for bacterial enzymes and as structural components of proteins. Both suboptimal and toxic concentrations of intracellular metal ions have severe effects on cellular metabolism. Under metal-limiting conditions, metal ions are internalised by active transport, whereas under conditions of metal excess, efflux or sequestration systems are induced. Hitherto, metal sensing in bacteria was believed to be accomplished exclusively by metal-responsive transcriptional regulator proteins. Based on structural studies, these proteins were classified into five families that bind a wide range of metal ions directly [10], yielding enhanced or decreased operator binding affinity or alteration of promoter structures. Among these, the DtxR, Fur and NikR families regulate genes encoding metal ion uptake proteins that – under metal-replete conditions – function as co-repressors to shut off transcription (Figure 1a). The iron(II)-dependent repressors DtxR, IdeR and Fur bind as dimers to palindromic sequences, thereby negatively regulating genes required for iron acquisition and virulence [11]. X-ray crystallographic structural studies have

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Table 1. Currently known riboswitches Regulated genes Riboflavin biosynthesis

Ligand FMN

Peculiarity trc/trl

Representative bacteria a, b, g proteobacteria Bacillales, Lactobacillales, Clostridiales Actinomycetales, Thermotogales

Thiamine synthesis, phosphorylation and transport

TPP

trc/trl most widespread

a, b, g, d, e proteobacteria Bacillales, Lactobacillales, Clostridiales Actinomycetales, Thermotogales Fusobacterales, Spirochaetales, Archaea

Cobalamin synthesis and transport Cobalt transport Ribonucleotide reductas Glutamate fermentation Succinate fermentation Uncharacterised genes

Vitamin B12

trc/trl largest ligand

a proteobacteria

Lysine synthesis and transport Lysine catabolism

Lysine

trc/trl, k-turn

Methionine biosynthesis Cysteine biosynthesis Methionine recycling SAM synthesis Methylene THF reductase Metabolite transport Uncharacterised genes

SAM

trc/trl, mostly Gram+ species, k-turn, loopE

g proteobacteria d proteobacteria Bacillales Actinomycetales Clostridiales/Thermoanaerobacteriales Cyanobacteria Chlorobiales, Fusobacterales

Purine synthesis and transport

Adenine Guanine

Gene activation, gene repression, trc

g, d, e proteobacteria

Glycine catabolism and efflux

Glycine

2 aptamer domains, cooperative Gly binding, trc/trl

a, b, g, d, e proteobacteria Bacillales, Lactobacillales, Clostridiales

GlcN6P synthesis

GlcN6P

Ribozyme, trc

Deinococcales, Bacillales, Clostridiales, Lactobacillales, Fusobacterales

Mg2+ influx

Mg2+

Smallest ligand trc

Salmonella, E. coli, Klebsiella, Citrobacter Erwinia, Serratia, Yersinia

b proteobacteria g proteobacteria d proteobacteria Deinococcales Bacillales, Lactobacillales, Clostridiales etc. g proteobacteria Bacillales

Abbreviations: FMN, flavin mononucleotide; GlcN6P, glucosamine-6-phosphate; k-turn and loop E are structural RNA motifs, also found in other contexts [7,8,15]; TPP, thiamine pyrophosphate; trc, transcriptional riboswitch; trl, translational riboswitch found for this ligand; SAM, S-adenosyl-methionine.

identified two distinct DtxR/IdeR metal sites: site 1 stabilizes the dimer, thereby facilitating binding of a metal ion to site 2, the putative metalloregulatory site activating DNA binding. The mechanism of allosteric positive regulation of operator binding by IdeR appears to involve a movement of the DNA-binding domain relative to the dimerization domain, enabling the DNA helices to fit snugly into successive major grooves [12]. Mg2+, the most abundant divalent cation in biological systems, is essential for cell survival. Among all biologically relevant cations, Mg2+ has the smallest ionic and largest hydrated radius and highest charge density, creating an intriguing problem for transport into living cells. Relatively little is known about the mechanisms by which cells sense Mg2+ to keep its intracellular concentration within a narrow range. The best characterised bacterium in this respect is Salmonella typhimurium, in which three Mg2+ transporters have been found: CorA, the major Mg2+ transporter in eubacteria, which is expressed constitutively, and MgtA and MgtB, which are regulated by the PhoP/PhoQ two-component system and ensure Mg2+ influx www.sciencedirect.com

into the cell when its concentration drops to micromolar levels [13]. A riboswitch that uses a metal ion as ligand A recent and surprising observation is that the 50 untranslated region (UTR) of the mgtA gene of S. typhimurium contains a riboswitch that can directly sense cytoplasmic Mg2+ [14]. This is the first riboswitch to be discovered that uses a metal ion as ligand. The riboswitch determines the fate of transcription complexes elongating into the coding region of mgtA, a control event that lies downstream from the transcription initiation control exerted by the PhoP/PhoQ system that responds to extracytoplasmic Mg2+. Cromie et al. [14] discovered that the 50 leader region of mgtA can fold into two alternative structures depending on the intracellular concentration of Mg2+. Binding of Mg2+ induces premature termination of mgtA transcription or, alternatively, transcription pausing followed by RNA polymerase release from the template, whereas submicromolar Mg2+ concentrations fail to induce termination or pausing, and the unbound leader region

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Figure 1. Metal ion sensing by proteins or RNA. (a) Fe2+ sensing by the B. subtilis Fur (ferric uptake regulator) protein (based on [18]). Under iron-depleted conditions, Fur does not bind Fe2+ but contains tightly bound structural Zn2+ (not shown) and cannot repress transcription by binding to its operator (Fur box) located downstream from the dhb promoter (black and white rectangles, –35 box and –10 box, respectively). Under conditions of iron excess, Fur binds Fe2+ and represses transcription of the dhb operon by binding as a dimer to its palindromic 19 bp operator sequence (two black triangles). dhb, dihydroxybenzoate siderophore biosynthesis. (b) Mg2+ sensing by the Salmonella enterica Mg2+-dependent riboswitch (based on [15]). Under submicromolar cytosolic Mg2+ concentrations, the leader region of the mgtA-mRNA folds into a transcriptional anti-terminator (SL C), enabling synthesis of the Mg2+ importer MgtA. When cytosolic Mg2+ concentrations are high, binding of Mg2+ to two regions in the mgtA RNA leader induces an alternative fold, which disrupts the anti-terminator and facilitates formation of SL A and SL B and a downstream terminator, leading to premature transcription termination and shut-off of MgtA synthesis. SL, stem-loop structure.

instead adopts a folding that permits transcriptional readthrough and, consequently, MgtA synthesis (Figure 1b). Such dual regulation of gene expression by a two-component system and a riboswitch that both respond to the same signal – in this case Mg2+ concentration – but in different cell compartments has not been reported before. Metabolite-sensing riboswitches are most often the sole regulators of expression of downstream genes. In cases where concerted regulation by two components has been demonstrated, such as the E. coli trp operon or the rep gene of plasmid pIP501, one of the mediators is a transcriptional repressor (TrpG and CopR, respectively) and the other an RNA modulator (trp-tRNA and the antisense RNA, respectively) both acting in the same compartment to control transcription attenuation [3,1]. The properties of the novel Mg2+-sensing riboswitch have been investigated in some detail, and the sequence responsible for regulation has been narrowed down: two regions crucial for Mg2+ sensing were identified at around nucleotides 73–74 and between nucleotides 91 and 106. A singleround transcription assay demonstrated that transcription termination increased with increasing concentrations of Mg2+, thus indicating that the ion alone is indeed the ligand responsible for control. Furthermore, enzymatic and chemical probing revealed that binding of Mg2+ to the mgtA 50 UTR modified the RNA structure. The riboswitch core was shown to comprise two predicted stem-loop structures, SL A (Mg2+ sensor) and SL B, which form predominantly in the www.sciencedirect.com

presence of high Mg2+ concentrations, and one alternative stem loop, SL C, that dominates at low Mg2+ concentrations (Figure 1b). Phylogenetic comparisons indicated that the Mg2+-sensing mechanism might be valid for a small range of other Gram-negative bacteria. Can other ions act as ligands for riboswitches? The most important ions with relevance for RNA folding in vivo are Mg2+ and K+, both of which interact predominantly by electrostatic forces. In the 1970s, the unusual effectiveness of Mg2+ to stabilize tRNA tertiary structure was observed; later, Mg2+ was found to be essential for the folding and, often, the activity of ribozymes. Several studies examined the effects of Mg2+ and K+ or Na+ ions on the formation of loopE of 5S rRNA: the crystal structure showed Mg2+ in the major groove and in its absence, K+ or Na+ occupied some of the same locations [15]. Recently, allosteric ribozymes were engineered to respond to Mn2+, Fe2+, Co2+, Ni2+, Zn2+ and Cd2+ [16]. However, the five ribozyme classes found by in vitro selection could not discriminate among this collection of effectors. Interestingly, riboswitches that depend on divalent metal ions or cationic compounds have been predicted in B. subtilis [17], although only upstream of genes that are not functionally assigned. This finding, together with the data mentioned above, does not seem to exclude entirely the concept that other metal ions might act as riboswitch ligands.

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Concluding remarks At this point, it is still unknown how widespread the use of metal ion sensing riboswitches are among prokaryotes or whether the specific riboswitch reported by Cromie and colleagues [15] is a biological speciality. Following the discovery that RNA can sense large molecules, such as moving ribosomes or antisense RNAs, or low molecular weight compounds, such as small metabolites with many functional groups that can potentially interact, we finally arrive at a tiny metal ion and find that it can cause the same effect: shutting down gene expression by forcing RNA into an alternative fold. The Mg2+-dependent riboswitch not only increases the regulatory repertoire of RNA but also lends further support to the hypothesis of an ancient RNA world. Most probably, ancient RNA motifs that bound metabolites or metal ions controlled the function of ribozymes before proteins emerged in evolution. References 1 Brantl, S. (2002) Antisense-RNA regulation and RNA interference. Biochim. Biophys. Acta 1575, 15–25 2 Gottesman, S. (2005) Micros for microbes: non-coding regulatory RNAs in bacteria. Trends Genet. 21, 399–404 3 Yanofsky, C. (1981) Attenuation in the control of expression of bacterial operons. Nature 289, 751–758 4 Grundy, F. and Henkin, T.M. (2004) Regulation of gene expression by effectors that bind to RNA. Curr. Opin. Microbiol. 7, 126–131

5 Mironov, A.S. et al. (2002) Sensing small molecules by nascent RNA: a mechanism to control transcription in bacteria. Cell 111, 747–756 6 Winkler, W. et al. (2002) Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419, 952–956 7 Winkler, W.C. and Breaker, R.R. (2005) Regulation of bacterial gene expression by riboswitches. Annu. Rev. Microbiol. 59, 487–517 8 Winkler, W.C. (2005) Riboswitches and the role of noncoding RNAs in bacterial metabolic control. Curr. Opin. Chem. Biol. 9, 594–602 9 Brantl, S. (2004) Bacterial gene regulation: from transcription attenuation to riboswitches and ribozymes. Trends Microbiol. 12, 473–475 10 Pennella, M.A. and Giedroc, D.P. (2005) Structural determinants of metal selectivity in prokaryotic metal-responsive transcriptional regulators. Biometals 18, 413–428 11 Andrews, S.C. et al. (2003) Bacterial iron homeostasis. FEMS Microbiol. Rev. 27, 215–237 12 Pohl, E. et al. (1999) Crystal structure of the iron-dependent regulator (IdeR) from Mycobacterium tuberculosis shows both metal-binding sites fully occupied. J. Mol. Biol. 285, 1145–1156 13 Moncrief, M.B. and Maguire, M.E. (1999) Magnesium transport in prokaryotes. J. Biol. Inorg. Chem. 4, 523–527 14 Cromie, M.J. et al. (2006) An RNA sensor for intracellular Mg2+. Cell 125, 71–84 15 Draper, D.E. et al. (2005) Ions and RNA folding. Annu. Rev. Biophys. Biomol. Struct. 34, 221–243 16 Zivarts, M. et al. (2005) Engineered allosteric ribozymes that respond to specific divaent metal ions. Nucleic Acids Res. 33, 622–631 17 Barrick et al. (2004) New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control. Proc. Natl. Acad. Sci. U. S. A. 101, 6421–6426 18 Moore, C.M. and Helmann, J.D. (2005) Metal ion homeostasis in Bacillus subtilis. Curr. Opin. Microbiol. 8, 188–195

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