Mg 2+ facilitates leader peptide translation to induce riboswitch-mediated transcription termination
2011; Springer Nature; Volume: 30; Issue: 8 Linguagem: Inglês
10.1038/emboj.2011.66
ISSN1460-2075
AutoresGuang Zhao, Wei Kong, Natasha Weatherspoon‐Griffin, Josephine E. Clark‐Curtiss, Yixin Shi,
Tópico(s)RNA modifications and cancer
ResumoArticle11 March 2011free access Mg2+ facilitates leader peptide translation to induce riboswitch-mediated transcription termination Guang Zhao Guang Zhao The School of Life Sciences, Arizona State University, Tempe, AZ, USA Search for more papers by this author Wei Kong Wei Kong The Center for Infectious Diseases and Vaccinology at the Biodesign Institute, Arizona State University, Tempe, AZ, USA Search for more papers by this author Natasha Weatherspoon-Griffin Natasha Weatherspoon-Griffin The School of Life Sciences, Arizona State University, Tempe, AZ, USA Search for more papers by this author Josephine Clark-Curtiss Josephine Clark-Curtiss The School of Life Sciences, Arizona State University, Tempe, AZ, USA The Center for Infectious Diseases and Vaccinology at the Biodesign Institute, Arizona State University, Tempe, AZ, USA Search for more papers by this author Yixin Shi Corresponding Author Yixin Shi The School of Life Sciences, Arizona State University, Tempe, AZ, USA The Center for Infectious Diseases and Vaccinology at the Biodesign Institute, Arizona State University, Tempe, AZ, USA Search for more papers by this author Guang Zhao Guang Zhao The School of Life Sciences, Arizona State University, Tempe, AZ, USA Search for more papers by this author Wei Kong Wei Kong The Center for Infectious Diseases and Vaccinology at the Biodesign Institute, Arizona State University, Tempe, AZ, USA Search for more papers by this author Natasha Weatherspoon-Griffin Natasha Weatherspoon-Griffin The School of Life Sciences, Arizona State University, Tempe, AZ, USA Search for more papers by this author Josephine Clark-Curtiss Josephine Clark-Curtiss The School of Life Sciences, Arizona State University, Tempe, AZ, USA The Center for Infectious Diseases and Vaccinology at the Biodesign Institute, Arizona State University, Tempe, AZ, USA Search for more papers by this author Yixin Shi Corresponding Author Yixin Shi The School of Life Sciences, Arizona State University, Tempe, AZ, USA The Center for Infectious Diseases and Vaccinology at the Biodesign Institute, Arizona State University, Tempe, AZ, USA Search for more papers by this author Author Information Guang Zhao1,‡, Wei Kong2,‡, Natasha Weatherspoon-Griffin1, Josephine Clark-Curtiss1,2 and Yixin Shi 1,2 1The School of Life Sciences, Arizona State University, Tempe, AZ, USA 2The Center for Infectious Diseases and Vaccinology at the Biodesign Institute, Arizona State University, Tempe, AZ, USA ‡These authors contributed equally to this work *Corresponding author. Corresponding author. School of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287-4501, USA. Tel.: +1 480 965 3904, Fax: +1 480 965 6899; E-mail: [email protected] The EMBO Journal (2011)30:1485-1496https://doi.org/10.1038/emboj.2011.66 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have characterized a 17-residue peptide, MgtL, which is translated specifically in high Mg2+ from an open reading frame (ORF) embedded in the Mg2+ riboswitch domain, previously identified in the 5′ leader region of Mg2+ transporter gene mgtA in Salmonella. We demonstrate that mgtL translation is required to prematurely terminate mgtA transcription. Abrogation of mgtL translation by mutation of its start codon results in transcription of the mgtA-coding region in high Mg2+, suggesting that ribosome stalling is not required for preventing premature transcription termination. Consistently, the Mg2+ riboswitch responds to cytoplasmic Mg2+, but not to proline or arginine, both repeatedly present in the MgtL sequence, to mediate mgtL translation-coupled regulation. RNA structural probing and nucleotide substitution analysis show that the riboswitch loop A region alters base pairing in response to Mg2+, and favours stem-loop A1 in high Mg2+, subsequently opening the ribosome-binding sequence for mgtL translation. Presumably, mgtL ORF directs translation to localize a ribosome in cis to act on downstream RNA in a manner similar to some upstream ORFs in prokaryotes and eukaryotes. Introduction In Mg2+-depleted conditions, bacteria facilitate uptake of this divalent cation by inducing synthesis of specific Mg2+ transporters (Snavely et al, 1991). The mgtA gene in the Gram-negative bacteria Salmonella typhimurium and Escherichia coli, which encodes a P-type ATPase to mediate Mg2+ influx (review see ref. Moncrief and Maguire (1999)), has served as a prototype in studies of both inducible regulation and biochemical function of genetic loci encoding Mg2+ transporters. It is suggested that two independent mechanisms are involved in Mg2+-dependent transcriptional regulation of mgtA. (i) The PhoP/PhoQ two-component system responds to micro-molar levels of environmental Mg2+ and activates transcription initiation; or to milli-molar levels of Mg2+ and represses transcription initiation (Garcia Vescovi et al, 1996). (ii) Once transcription is initiated, the 5′ untranslated region (here, called the 5′ leader region or 5′LR) of nascent mgtA transcripts functions as an alternative Mg2+-sensing system. If the Mg2+ concentration increases in the bacterial cytoplasm, the latter system interrupts mgtA transcription before it is extended to the downstream coding region (Cromie et al, 2006). In Salmonella, the mgtA transcript initiated from the QJ;PhoP-activated promoter contains a 264-nt 5′LR (Lejona et al, 2003), which contains a cis-acting regulatory element responsive to Mg2+. This element is similar to other riboswitches that are able to interact with a small molecule, normally a specific metabolite, and modify the RNA structures through stem-loop switching, subsequently exerting their regulatory effects (review see ref. Tucker and Breaker (2005)). A structural probing of the 5′LR showed that a high Mg2+ condition (3.5 mM) induced regions containing nucleotides 56–125 and 136–159 to form stem-loops A and B (see ref. Cromie et al (2006) and illustrated in Figure 1A). Whereas, a low Mg2+ condition (0.35 mM) caused a stem-loop switching via alternative base pairing between nucleotides 118–125 located in the right arm of stem A, and 140–147 in the left half of stem-loop B, resulting in formation of stem-loop C. Stem-loop B is a prerequisite for initiating premature termination of mgtA transcription mediated by the mgtA Mg2+ riboswitch (Cromie et al, 2006). The stem-loop C, favoured in low Mg2+, may have prevented formation of stem-loop B, and thus allowed mgtA transcription to be extended into the coding region (Cromie et al, 2006). The E. coli mgtA 5′LR also responds to Mg2+ similarly as its Salmonella homologue (Cromie et al, 2006). A truncated RNA representing the mgtA 5′LR was characterized from E. coli cells (Kawano et al, 2005), providing direct evidence for premature termination of mgtA transcription in vivo. Notably, the truncated transcripts are different in length from in vivo (∼240-nt; Kawano et al, 2005) and in vitro (220-nt; Cromie et al, 2006) samples. As the mgtA 5′LR does not have sequences consistent with a Rho-independent terminator, the 220-nt transcript is unlikely a product generated in vitro through transcription termination, but a product from the strong pausing of the RNA polymerase in high Mg2+. The mechanism of termination or pausing, however, is not known. It is possible that mgtA transcription is paused at nucleotide 220, probably by an RNA conformation induced in high Mg2+, and subsequently terminated near nucleotide 240 in vivo by additional cellular components. Figure 1.Prediction of the MgtL leader peptide encoded by the 5′LR of mgtA homologues in Gram-negative bacteria. (A) A schematic representation of the Mg2+ riboswitch domain in Salmonella mgtA 5′LR (5′LR). Coloured letters are the nucleotide sequences involved in the stem-loop switching in different Mg2+ concentrations. Numbering represents the positions of nucleotides in the mgtA 5′LR. Uppercase letters are the stem-loop structures formed in different Mg2+ concentrations. Highlighted sequences are the start and stop codons of the mgtL open reading frame (ORF). The dotted lines indicate that base pairing is possible, however, our data does not support it. (B) Sequence alignment of the stem-loops A and B in the 5′LR region of the mgtA gene. Sequences in colour and underlined in red are stem-loop structures A and B, and the ribosome-binding sites (SD). Three-letter sequences black framed, and underlined in black are start and stop codons, and codons in the mgtL ORF. Orange and green frames are the sequences to form stem A1. Amino-acid residues in the MgtL peptide from Salmonella are shown. Numbering represents the positions of nucleotides in the mgtA 5′LR. (C) MgtL peptides predicted from Gram-negative bacterial species. Highlighted residues are conserved in these MgtL peptide sequences. Download figure Download PowerPoint Stem-loop A is critical for Mg2+ sensing in the riboswitch because Mg2+-promoted conformational changes in stem-loops B and C depend on the presence of the stem-loop A sequence (Cromie et al, 2006). As the stem-loop A region is transcribed, it might trap the 5′LR RNA into distinct structures depending on the Mg2+ concentration that ultimately determine whether transcription is prematurely terminated. Interestingly, a transition mutation in loop A, that substituted nucleotide 98 from C to U, resulted in uncharacteristic expression of Salmonella mgtA in high Mg2+ concentrations (O'Connor et al, 2009). While the significance of stem-loop A has been implicated, it remained unknown what regulatory element embedded in this region contributed to the 5′LR function. Importantly, previous results suggested that additional cellular factors could have a role in transcriptional regulation of mgtA via the 5′LR. (i) When transcribed in E. coli, mgtA transcript from Salmonella was degraded more in a high Mg2+ condition in an RNase E-dependent manner (Spinelli et al, 2008). Mutations at the 5′LR eliminated the degradation, suggesting that this nuclease degrades the mgtA mRNA by targeting the 5′LR. (ii) A transcriptional regulator, Rob, can bind to the Salmonella sequence: 5′-accgccaTaattgccacaaa-3′, which includes the PhoP-dependent transcription start (shown in uppercase) (Barchiesi et al, 2008). When overexpressed, Rob initiates transcription from nucleotide 44 of the 5′LR in a PhoP/PhoQ-independent manner. An Mg2+-responsive RNA element was also characterized in Gram-positive bacteria. The 5′LR of the Mg2+ transporter gene, mgtE from Bacillus subtilis, harbours a metal-sensing domain (M box), which is able to bind Mg2+ and enhance formation of the downstream Rho-independent terminator structure (Dann et al, 2007). Interestingly, these regulatory RNAs from Gram-negative and Gram-positive bacteria do not share homologous sequences, suggesting that they employ different mechanisms to sense Mg2+ and mediate transcription regulation. In this study, we identify a novel component that controls the regulatory function of the Mg2+ riboswitch in mgtA. Our results demonstrate that the stem-loop A region in the 5′LR comprises a translational unit, which encodes the 17-residue peptide, MgtL, in Salmonella. We show that Mg2+ facilitates modification of the stem-loop A conformation through stem switching to allow mgtL translation from the integral open reading frame (ORF), resulting in premature termination of mgtA transcription. This mechanism seems to be adopted by the MgtA-type Mg2+ transporters in many other Gram-negative species, providing an example in which a small molecule ligand stimulates regulatory function of its cognate riboswitch to initiate premature termination of transcription by coupling translation of a leader peptide. During the submission of our manuscript, a publication became available online which reported an ORF encoding a putative peptide MgtL in the Salmonella mgtA 5′LR presumably responsive to proline (Park et al, 2010). While the presence of the mgtL ORF is undisputed, our model of the Mg2+-dependent/proline-independent mgtL translation via a novel stem-loop switch does not support their conclusions. Results An ORF embedded in the stem-loop A region of the mgtA 5′LR To study the regulatory function of stem-loop A, we analysed the phylogenetically conserved sequences from several Gram-negative species that harbour mgtA homologues, including S. typhimurium, E. coli, Klebsiella pneumoniae, Citrobacter rodentium, Erwinia chrysanthemi, Serratia marcescens, and Yersinia enterocolitica. These stem-loop A sequences greatly varied, but contained three highly conserved regions that could form an ORF: (i) a consensus sequence for ribosome-binding (SD) located upstream of a start codon, (ii) a start codon, AUG (GUG in Yersinia), and (iii) a stop codon, UAA, lying in the same reading frame (Figure 1B). In Salmonella, this ORF encodes a 17-residue peptide (referred to as MgtL, hereafter) from the start codon 71AUG73, which is located 4-nt downstream of a putative SD sequence 62GGAGG66, to the stop codon 122UAA124 (Figure 1B). MgtL homologues can also be predicted from stem-loop A sequences of other Gram-negative species (Figure 1B). MgtL in E. coli, K. pneumoniae, and C. rodentium are also 17-residue peptides sharing high identity with that in Salmonella (70.6, 76.5, and 64.7%, respectively, Figure 1C). On the other hand, MgtL from E. chrysanthemi, S. marcescens, and Y. enterocolitica are shorter peptides, and merely share proline residues at positions 3 and 5 (amino acid residues highlighted in Figure 1C), and arginine at the C terminus, as with the peptides from other species. Regardless of the varied sequence and length in these species, the stop codon UAA is always located at the end of the right arm of stem A (Figure 1B, except Y. enterocolitica whose UAA is located just before the right arm). The right arm is the switching sequence in the riboswitch structure, that base pairs with alternative sequences to form stem-loop C in low Mg2+ and stem-loop A in high Mg2+ (Cromie et al, 2006). We presume that this architectural design of the mgtL ORF is important for the regulatory function of the mgtA 5′LR. Similar to our observation, an 18 codon ORF, predicted to encode a peptide whose suggested sequence is the same as MgtL, was identified from Salmonella mgtA 5′LR in a recent study (Park et al, 2010). Characterization of MgtL peptide encoded by the stem-loop A sequence in Salmonella mgtA 5′LR The MgtL peptide is probably either highly unstable or produced at very low levels in the conditions used in this study. We were unable to detect MgtL peptide expressed from the 5′LR in the chromosomal location in vivo using western blot. Therefore, we constructed a plasmid, pYS1475, which carries the full-length mgtA 5′LR with an inserted 21-nt sequence encoding the FLAG-epitope to generate MgtL tagged by FLAG at the N terminus (hereafter MgtL–FLAG; Figure 2A). In this plasmid, the Plac1−6 promoter (Liu et al, 2004), which is independent of Mg2+ and the PhoP/PhoQ system (Cromie et al, 2006; Kong et al, 2008), initiates transcription of the 5′LR and a downstream lacZ gene. Notably, the Rob regulator does not control this transcription because the Rob-binding site is partially deleted in this plasmid (data not shown). β-Galactosidase activity in wild-type Salmonella harbouring pYS1475 and its parent plasmid pYS1010 (i.e., Plac1−6-mgtA 5′LR-lacZ) (Cromie et al, 2006) grown in N minimal medium (Snavely et al, 1989) supplemented with 0.01 mM (low) Mg2+ are 13.3- and 18-fold higher than those with 10 mM (high) Mg2+, respectively. This suggests that the engineered mgtA 5′LR responds similarly to Mg2+ as wild-type 5′LR. Because the Salmonella 5′LR can also function in E. coli (β-galactosidase activity from MC4100 harbouring pYS1010 grown in low Mg2+ is ∼15-fold higher than in high Mg2+), we introduced pYS1475 into an E. coli Maxicell mutant, CSR603. MgtL–FLAG was produced in UV-irradiated bacterial cells in which protein synthesis directed by chromosomal loci, but not by plasmid, was generally inhibited due to extensive degradation of the chromosomal DNA (Sancar et al, 1979). Affinity chromatography was carried out to isolate MgtL–FLAG (MW 3164 da) from bacterial cultures grown in low and high Mg2+. The peptide sample was separated through electrophoresis and a band was detected from the bacterial cells grown in high Mg2+ (Figure 2B), which migrated to a position slightly slower than a control peptide, magainin 2 (MW 2465 da). However, this peptide could not be detected from the bacterial cells grown in low Mg2+, suggesting that MgtL–FLAG is synthesized only in high Mg2+. We then carried out a parallel experiment using a plasmid, pYS1475–A71C, which carries an A–C substitution at nucleotide 71 of the 5′LR that changes 71AUG73 to 71CUG73, resulting in deletion of the start codon. The MgtL–FLAG peptide could not be detected from the cells harbouring this plasmid grown in low and high Mg2+ (Figure 2B). Furthermore, when MgtL was overexpressed in a Salmonella wild type harbouring an IPTG-inducible plasmid pUHE-mgtL, we were able to detect an m/z 2171.38 peak in a MALDI-TOF mass spectrum analysis from an eluent derived from bacteria cells grown in the presence of IPTG and 10 mM Mg2+ (Figure 2C, bottom). This peak is specific because it could not be detected from a wild-type cell lysate harbouring control vector (Figure 2C, top). Figure 2.Characterization of the MgtL leader peptide encoded by the 5′LR of Salmonella mgtA gene. (A) A schematic representation of the FLAG insertion site in plasmid pYS1475 containing Salmonella mgtA 5′LR. A site-directed substitution is marked in the small frame, and (1) is the mutated start codon in the MgtL sequence. (B) Silver staining of Salmonella MgtL peptides. Peptide preparations were derived from E. coli Maxicell mutant (CSR603) harbouring pYS1475 and pYS1475-A71C. Bacterial cultures were subjected to UV irradiation (50 J/m2) for 2, 3, 5, and 10 min to enhance MgtL–FLAG synthesis. H and L represent N medium supplemented with 10 and 0.01 mM Mg2+, respectively. Arrow indicates the position of magainin 2. Asterisk represents MgtL–FLAG bands. (C) MALDI-TOF mass spectrum analysis of MgtL from Salmonella harbouring vector pUHE (top) and pUHE-mgtL (bottom) grown for 4 h in N medium with 0.5 mM IPTG. m/z represents the mass-to-charge ratio, and MgtL peptides carry one positive charge. Download figure Download PowerPoint Premature termination of Salmonella mgtA transcription in high Mg2+ is coupled to mgtL translation initiation The observation that mgtL translation and premature termination of mgtA transcription both occur in the 5′LR in high Mg2+ suggests that these convergent phenomena are coordinated in response to Mg2+. Thus, we hypothesize that MgtL synthesis is a prerequisite for the premature termination of mgtA transcription. We constructed a set of pYS1010 derivatives with site-directed substitutions inside the mgtL-coding region (Figure 3A and C), and determined lacZ expression in Salmonella wild-type cells harbouring these plasmids. In contrast to the result from parental pYS1010, lacZ expression in cells carrying pYS1010-A71C, in which MgtL could not be synthesized due to disruption of the start codon (Figure 2A and B) remained activated in high Mg2+ because β-galactosidase activity was only 1.9-fold lower in high Mg2+ than in low Mg2+ (Figure 3B). On the other hand, lacZ transcription from cells harbouring pYS1010-A71G, which also carried a substitution at nucleotide 71, but changed 71AUG73 to another start codon 71GUG73 (Figure 3A and C), was repressed in high Mg2+ because β-galactosidase activity was 12.2-fold lower when grown in high Mg2+ than in low Mg2+ (Figure 3B). To further determine the importance of the start codon, we tested pYS1010-G73C in which the start codon was disrupted by a substitution at nucleotide 73 (Figure 3A and C). Comparable to pYS1010-A71C, β-galactosidase activity from cells with pYS1010-G73C was only two-fold lower in high Mg2+ than in low Mg2+ (Figure 3B). Apparently, if mgtL fails to be translated, high Mg2+ is not sufficient to prematurely terminate mgtA transcription. The 5′LR in another plasmid, pYS1010-G74C, which contains a substitution at nucleotide G74 that changes the second amino acid from Asp to His without interfering with mgtL translation (Figure 3A and C) remained responsive to Mg2+ because β-galactosidase activity in high Mg2+ was 14.9-fold lower than in low Mg2+ (Figure 3B). Collectively, these observations demonstrate that mgtL translation is essential for the premature termination of mgtA transcription in high Mg2+. Figure 3.Genetic evidence demonstrates that MgtL translation is coupled to premature termination of mgtA transcription in the mgtA 5′LR. (A) Illustration of the substituted nucleotides in pYS1010 derivatives. Numbering represents the positions of the nucleotides in the Salmonella mgtA 5′LR. Framed sequences represent the start and stop codons. The SD sequence is underlined. (B) β-Galactosidase activity was determined in Salmonella wild-type 14028s, which harboured wild-type plasmid, pYS1010, or one of the substituted derivatives shown in (A). Bacteria were grown for 4 h in N medium supplemented with 0.01 mM (low) or 10 mM (high) Mg2+. Fold change was determined by β-galactosidase activity from low Mg2+ divided by activity from high Mg2+. Assays were conducted in triplicate. Error bars correspond to the standard deviation. (C) Effect of the substitutions of the mgtL ORF on premature termination of mgtA transcription based on the results from (B). Download figure Download PowerPoint Disruption of mgtL translation elongation prevents the premature termination of mgtA transcription in high Mg2+ We created a stop codon within the mgtL ORF to determine whether interference of its translation elongation could inhibit premature termination of mgtA transcription in high Mg2+. The plasmid, pYS1010-G80T, harbours a substitution that replaces the fourth codon, 80GAA82 (Glu), with stop codon 80UAA82 (Figure 3A and C), in which mgtL translation should be stopped prematurely. β-Galactosidase activity from cells harbouring this plasmid in high Mg2+ was only 1.4-fold lower than that in low Mg2+ (Figure 3B), indicating that mgtA transcription could not be prematurely terminated due to the nonsense point mutation. Furthermore, a recent study showed that a substitution, C98U, in the mgtA 5′LR resulted in mgtA expression in high Mg2+ (O'Connor et al, 2009). This mutation changes the tenth codon, 98CGA100 (Arg), to a stop codon 98UGA100, thus causing a premature stop of mgtL translation at a codon far downstream of 80GAA82 (fourth codon). We constructed a plasmid pYS1010-C98A, which carried a substitution at the same nucleotide, C98, and generated a silent mutation (Figure 3A and C) and found that β-galactosidase activity was 27.9-fold lower in high Mg2+ than in low Mg2+ (Figure 3B), indicating that the 5′LR carrying a substitution of C98A, unlike C98U, remained responsive to Mg2+. With these results and the observation that the full-length MgtL peptide is detected specifically in high Mg2+ (Figure 2B), we propose that mgtL translation should be completed in high Mg2+ to prematurely terminate mgtA transcription. Mg2+ concentration modulates a stem switching within stem-loop A that determines conformation of the ribosome-binding site for mgtL translation We synthesized the full-length 264-nt mgtA 5′LR and probed the stem-loop A structure in different Mg2+ conditions using dimethyl sulphate (DMS) which modifies adenosine, cytidine, and guanosine when located in single-stranded regions. A primer extension assay, in which the reverse transcription reaction is disrupted at the modified nucleotides in RNA templates, showed that 62GGAGG66, proposed to be the SD sequence here (Figure 1B), was located in a double-stranded region in low Mg2+, however, in a single-stranded region in high Mg2+ (Figure 4A). The nucleotides G63 and A64 in the SD sequence were modified 2.7- and 2.4-fold more in high Mg2+ (3 mM) than in low Mg2+ (0.1 mM), respectively (Figure 4A), indicating their locations in a single-stranded region in high Mg2+ regardless of simulated base pairs (Cromie et al, 2006). Furthermore, G73, G74, C85, A86, C96, and G97 were modified 2.9-, 2.4-, 2.6-, 4.7-, 2.1-, and 2.6-fold more in high Mg2+ than in low Mg2+, respectively, implying that they are base paired or protected in a stem (named D, Figure 1A) formed in low Mg2+. Based on these observations, we proposed that the sequence, 91UCUCC95 (named anti-SD), can form part of stem D by base pairing with the SD sequence in low Mg2+; and can alternatively form stem A1 with the sequence, 102GGAGA106 (named anti-anti-SD), in high Mg2+ in which the SD site is accessible for mgtL translation (summarized in Figure 6). Consistent with this, substitution of the anti-SD sequence in stem A1 with 91AGAGG95 enhanced premature transcription termination regardless of Mg2+ because β-galactosidase activity in a wild-type strain harbouring this substituted plasmid (pYS1010-A1-sub) grown in low Mg2+ was as low as that in wild-type strain harbouring the wild-type plasmid grown in high Mg2+ (Figure 4B, also see ref. Cromie et al (2006)). To determine the role of the anti-SD sequence, we used DMS to map the full-length RNA carrying this substituted sequence. We found that G63 and A64 in the SD sequence was modified regardless of Mg2+ at a similar level as wild type in high Mg2+ (Figure 4C), indicating that without the anti-SD sequence, the SD sequence remained single stranded in low and high Mg2+, causing constitutive repression of mgtA transcription likely due to the continuous translation of mgtL. Introduction of a second substitution to pYS1010-A1-sub, which replaced 102GGAGA106 with 102CCUCU106 to form pYS1010-A1-rev, complemented the first substitution by creating a modified stem-loop A1 and restored a wild-type-like response to Mg2+ (Figure 4B, also see ref. Cromie et al (2006)). It is likely that 102CCUCU106 forms a new anti-SD sequence that base pairs with the SD sequence, thus resulting in inhibition of mgtL translation similar to wild type in low Mg2+; whereas the 91AGAGG95 becomes a new anti-anti-SD sequence in the double substituted 5′LR and turns on mgtL translation in high Mg2+. This was supported by a DMS probing assay using this double substituted full-length RNA in which, like wild-type RNA, the nucleotides G63 and A64 in the SD sequence were protected from DMS modification in low Mg2+ (Figure 4C). Additional mapping of the full-length wild-type RNA with RNase T1, which cleaves unpaired G residues, revealed that high Mg2+ facilitates the accessibility of this nuclease to G65 and G66 located in the SD sequence because they were cleaved 3.4-fold more in high Mg2+ than in low Mg2+ (Supplementary Figure S2), suggesting that the SD site was localized in a single-stranded region in high Mg2+ making it more accessible. In contrast, G105 in the anti-anti-SD sequence was cleaved 3.7-fold more in low Mg2+ than in high Mg2+, implying that it should be located in double-stranded region by base paring with the anti-SD sequence in high Mg2+, however, located in a single-stranded region when the anti-SD sequence is switched to form stem-loop D in low Mg2+ (Figure 6). Collectively, these results provide evidence that Mg2+ controls the accessibility of the mgtL SD sequence via a stem-loop switching that determines formation of stem-loop A1 and D by which it modulates mgtL translation. Figure 4.Mg2+ modifies the secondary structure of stem-loop A in the full-length 264-nt mgtA 5′LR. (A) Primer extension of DMS-treated stem-loop A following incubation with 0.1, 0.3, 1, and 3 mM Mg2+. In both (A) and (C), 6% polyacrylamide gel was used to separate the products. Lane M corresponds to Maxam–Gilbert reaction using DNA fragment amplified from pYS1010 with primers 220 and 32P-labeled 201. Lane C corresponds to a reaction with untreated RNA. Quantification in both (A) and (C) was conducted using Quantity One software (Bio-Rad). After all bands in a lane were normalized by an unchanged band at different Mg2+, the DMS modification ratio was calculated and shown on the right of (A) by comparing with the corresponding band from the sample incubated with 0.1 mM Mg2+. The x-axis represents the DMS modification ratio at a nucleotide as calculated while its position is shown on the y-axis. (B) The mgtA 5′LR with substitution at 91–95 does not respond to Mg2+ and the transcription is significantly reduced. The SD and stem-loop A1 sequences of plasmid pYS1010 and derivatives with the substitutions are shown. Numbering represents position of nucleotide in the mgtA 5′LR. β-Galactosidase activity was determined in Salmonella wild-type 14028s, which harboured wild-type plasmid pYS1010 or derivatives. Bacteria were grown for 4 h in N medium supplemented with 0.01 mM (low) or 10 mM (high) Mg2+. (C) DMS modification of the full-length 264-nt 5′LR with wild-type or substituted sequences shown in (B). The RNA was incubated with 0.1 mM (L) and 3 mM (H) Mg2+ before DMS treatment and primer extension. After all bands in a lane were normalized by an unchanged band at different Mg2+, the DMS modification ratio was calculated and shown on the bottom of (C) by comparing with the corresponding band from wild-type sample incubated with 0.1 mM Mg2+. Download figure Download PowerPoint Similar stem-switching domains that determ
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