Erschienen in: Microbiological Research ; 195 (2017). - S. 71-80

https://dx.doi.org/10.1016/j.micres.2016.11.003

TPP characterization in tabrizica and

Alishewanella aestuarii and comparison with other TPP

a,b,c d a e,f

Elnaz Mehdizadeh Aghdam , Malte Sinn , Vahideh Tarhriz , Abolfazl Barzegar ,

d,∗∗ a,b,∗

Jörg S. Hartig , Mohammad Saeid Hejazi

a

Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

b

Molecular Medicine Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

c

Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran

d

Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany

e

Research Institute for Fundamental Sciences (RIFS), University of Tabriz, Tabriz, Iran

f

The School of Advanced Biomedical Sciences (SABS), Tabriz University of Medical Sciences, Tabriz, Iran

a b s t r a c t

Riboswitches are located in non-coding areas of mRNAs and act as sensors of cellular small molecules,

regulating expression in response to ligand binding. The TPP riboswitch is the most widespread

riboswitch occurring in all three domains of life. However, it has been rarely characterized in environ-

mental other than Escherichia coli and Bacillus subtilis. In this study, TPP riboswitches located



in the 5 UTR of thiC from Alishewanella tabrizica and Alishewanella aestuarii were identified and

characterized. Moreover, affinity analysis of TPP binding to the TPP aptamer domains originated from

A. tabrizica, A. aestuarii, E.coli, and B. subtilis were studied and compared using In-line probing and Sur-

face Plasmon Resonance (SPR). TPP binding to the studied from A. tabrizica and A. aestuarii caused

distinctive changes of the In-line cleavage pattern, demonstrating them as functional TPP riboswitches.

With dissociation constant of 2–4 nM (depending on the method utilized), the affinity of TPP binding was

highest in A. tabrizica, followed by the motifs sourced from A. aestuarii, E. coli, and B. subtilis. The observed

variation in their TPP-binding affinity might be associated with adaptation to the different environments

of the studied bacteria.

1. Introduction lar ligands (Nahvi et al., 2002). The organization of riboswitches

usually consists of an aptamer and an expression platform.

Several gene regulation mechanisms have been established to Specific binding of the corresponding ligand to the aptamer domain

adapt the cells to various changing conditions. However, RNA has causes a conformational change in the expression platform lead-

been shown to play a significant role in controlling gene expres- ing to the creation of a transcription terminator structure and/or

sion. RNA regulatory elements are usually divided into trans-acting masking of the ribosomal binding site (RBS) to prevent translation

riboregulators such as microRNAs (miRNA) and short interfering initiation or vice versa (Serganov and Patel, 2012). Until now about

RNAs (siRNA) and cis-acting regulating RNAs within non-coding 30 different classes of riboswitches have been identified. How-

parts of RNA (reviewed in Waters and Storz, 2009). Riboswitches, ever, many candidate riboswitches identified by bioinformatics still



as one of the cis-acting type elements, are usually located in 5 UTRs await ligand-assignment (Weinberg et al., 2007, 2010).

of mRNAs where they regulate the transcription and/or translation pyrophosphate (TPP) is the active form of vitamin B1

of the respective gene through specific binding to small molecu- and an important factor in several metabolic pathways (Vander

Horn et al., 1993). The TPP riboswitch is the most widespread

riboswitch and was also one of the first riboswitches introduced

by Breaker and co-workers in E. coli (Winkler et al., 2002) and

∗

Corresponding author at: Dept. of Pharmaceutical Biotechnology, Faculty of

Mironov et al. in B. subtilis (Mironov et al., 2002). In E. coli the UTR

Pharmacy, Tabriz University of Medical Sciences, Tabriz 51664, Iran.

∗∗ upstream of the thiM and thiC open reading frames were studied

Corresponding author at: University of Konstanz, Department of Chemistry,

Room L904, Mailbox A704, Universitätsstr. 10, 78457 Konstanz, Germany. and conserved parts of the aptamer domain were observed. The

E-mail addresses: [email protected] (J.S. Hartig), mechanism of gene regulation in thiM RNA was suggested to be

[email protected], [email protected] (M.S. Hejazi).

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-394912

72

Shine Dalgarno-masking. However, the riboswitch in thiC is respon- 2. Materials and methods

sible for controlling by both SD-masking and

transcription attenuation. In Bacillus subtilis, the mRNA upstream 2.1. Preparation of RNA sequences from different bacteria

of the tenA ORF was studied and a thi-box was also identified

and transcription termination was shown to be controlled by TPP 2.1.1. Providing TPP riboswitch template for in vitro transcription

binding. Later, it was revealed that some representatives of TPP In order to prepare TPP riboswitches RNAs, first PCR amplifica-

riboswitch also exist in including fungi (in Aspergillus tions with corresponding primers (Table S1) were carried out. T7

oryzae thiA gene (Kubodera et al., 2003)), as well as plants (Oryza RNA polymerase promoter sequence was added upstream of the



sativa 3 UTR of a putative thiC gene (Sudarsan et al., 2003)), the TPP riboswitch aptamer domain. For RNAs to be used in SPR, for-

tomato LeTHIC transcript (Zhao et al., 2011), A. thaliana (Thore ward primers were designed to insert a linker of 21 bp between T7

et al., 2008), and the algae Chlamydomonas reinhardtii thi4 and RNA polymerase promoter sequence and TPP riboswitch.

thiC transcripts (Croft et al., 2007). The gene regulation mecha- The procedure to provide templates for each bacterium and PCR

nisms were studied for eukaryotic TPP riboswitches that controls details are explained below.

intron splicing (Kubodera et al., 2003) involving long distance

base pairing in Neurospora crassa (Li and Breaker, 2013). Structural 2.1.1.1. A. aestuarii TPP riboswitch. The genome of A. aes-

studies were carried out on the crystallographic structure of TPP tuarii is available under the accession number of RefSeq:

riboswitch in thiM mRNA (Serganov et al., 2006; Warner and Ferre- NZ ALAB00000000.1 (Jung et al., 2012). The sequence of thiC



D’Amare, 2014) and Arabidopsis thaliana (Thore et al., 2008). In operon containing 5 UTR was extracted from the genome and then

addition, the folding mechanism upon ligand binding was studied TPP riboswitch was confirmed RNAReg 2.0 scanning. The whole



at the single-molecule level using both force spectroscopy and sin- sequence of 5 UTR was synthesized by GeneArt Company (Table

gle molecule-Förster resonance energy transfer recently (smFRET) S1-oligo 1) and was cloned in pTZ57R/T plasmid using TA cloning kit

(Duesterberg et al., 2015). Moreover, the TPP aptamer has been used (Theremo scientific) (pTZ/AaesTPP). Constructed pTZ/AaesTPP was

to construct synthetic riboswitches utilizing ribozymes as expres- used as the template for PCR reaction. Besides, to obtain desired TPP

sion platform (Wieland et al., 2009). riboswitch aptamer domain for In-line probing and SPR, primers

TPP riboswitches in E. coli and B. subtilis were the first exam- 15/17 and 16/17 (Table S1) were employed, respectively,

ples of riboswitches as targets of antibiotics. In this case, the

ligand analog pyrithiamine was demonstrated to bind and trig- 2.1.1.2. A. tabrizica TPP riboswitch. Not being available the com-

ger the riboswitch, thereby shutting off the TPP biosynthesis plete genome of A. tabrizica, multiple alignment using Clustal



(Sudarsan et al., 2005). This discovery led to the general idea of Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) was done on 5

designing novel antibiotics based on the structure of riboswitches UTR and thiC gene of A. aestuarii, A. jeotgali and A. agri to identify

(Blount and Breaker, 2006). Consequently, some drug discovery conserved sequences for primer design. This resulted in design of

approaches were utilized to find TPP riboswitches targeting antibi- primers 8, 9 and 10 (Table S1). Then, in order to design a reverse

otics (Cressina et al., 2011; Warner and Ferre-D’Amare, 2014; primer based on A. tabrizica sequence, in the first round of PCR,

Warner et al., 2014). Owing to the potential of riboswitches in novel part of thiC gene was amplified using primers 8/9 and PCR product

antibiotic design and discovery, pathogenic bacteria are interest- was sequenced. Then, reverse primer 11 was designed based on

ing area to the study of riboswitches. However, environmental the sequenced area. The PCR reaction was then conducted using

microorganisms other than B. subtilis have been rarely used for primers 10/11 and the product was cloned into pTZ57R/T plas-

studying riboswitches. These bacteria due to their different con- mid (pTZ/AtabTPP), sequenced and the obtained sequence was

ditions as well as possibly diverse metabolic pathways could show confirmed as TPP riboswitch using RNAReg 2.0 scanning (http://



different patterns of gene regulation such as riboswitch-mediated regrna2.mbc.nctu.edu.tw/). The partial sequence of 5 UTR and

mechanisms. Here we studied the TPP riboswitches of two Alishe- thiC gene was submitted to NCBI under the accession number of

wanella species. KU886141. PCR reaction was carried out to obtain desired TPP

The genus Alishewanella from the phylum of ␥- riboswitch aptamer domain using pTZ/AtabTPP as the template

was first introduced in 2000 and the first bacterium in this genus, using primers 12/14 and 13/14 for In-line probing and SPR experi-

considering the close proximity to genus , was named ments, respectively.

Alishewanella fetalis (Vogel et al., 2000). About a decade later, other

species in this genus were identified including Alishewanella jeot- 2.1.1.3. E. coli and B. subtilis TPP riboswitch. In order to obtain TPP

gali (Kim et al., 2009), Alishewanella aestuarii (Roh et al., 2009) riboswitch of E. coli and B. subtilis, the annotated TPP riboswitches



and (Kim et al., 2010) isolated from traditional in 5 UTR of thiC gene of accession numbers CP014225.1 and

fermented food, tidal flat sediment, and landfill soil, respectively. CP014166.1 were amplified, respectively, using colony PCR. For

Alishewanella tabrizica, a Gram-negative, aerobic, motile and rod- E. coli, primers 5/7 and 6/7 (Table S1) and for B. subtilis primers 2/4

shaped bacterium was identified by our group in 2012 isolated from and 3/4 (Table S1) were employed to obtain TPP riboswitch aptamer

a Qurugöl Lake located in the north-west of Iran (Tarhriz et al., domain for In-line probing and SPR experiments, respectively.

2012). Alishewanella solinquinati is the last specie introduced in this

genus isolated from soil contaminated with textile dyes (Kolekar 2.1.1.4. Secondary structures. Secondary structures of TPP

et al., 2013). Apart from some annotations regarding the predic- riboswitches were predicted with Mfold (http://unafold.rna.

tion of cobalamin and c-di-GMP riboswitches (GeneBank accession albany.edu/?q=mfold) (Zuker, 2003).

number of LCWL01000001.1 and LCWL01000143.1, respectively)

no experimental identification or confirmation of riboswitches 2.1.2. In vitro transcription

have been carried out in this genus. In this study, we identify and Ethanol precipitated PCR products were employed as templates

characterize the TPP riboswitch of thiC operon in A. aestuarii and A. for in vitro transcription using a reaction containing T7 RNA poly-

tabrizica and compare these with TPP riboswitches in E. coli and B. merase, pyrophosphatase (ppase), RNase inhibitor and incubated

◦

subtilis. For this purpose, TPP binding was examined using In-line for 3 h at 37 C. DNA was removed by DNaseI treatment. RNA sam-

probing and surface plasmon resonance (SPR) methods to deter- ples were run in 8% denaturing PAGE (urea 9 M). Then, the desired

mine the affinity of TPP to these motifs. bands were cut out and RNAs were extracted from the gel using

73

crush soak buffer (10 mM HEPES, 200 mM NaCl, and 1 mM EDTA, exposed on a phosphorimager screen overnight and scanned using

pH 7.5). The eluted RNAs were precipitated with ethanol and resus- a Typhoon laserscanning system (GE Healthcare). Band volumes

pended in Mili-Q water. were determined using Quantity One software and accordingly Kd

(dissociation equilibrium constant) was calculated as described by

2.2. In-line probing Regulski and Breaker (Regulski and Breaker, 2008a) using following

equilibrium:

In-line probing was carried out based on the protocol described

samplevalue − min value

=

by Regulski and Breaker (Regulski and Breaker, 2008b) as follows. FractionRNAcleaved

max value − min value

The experiment was conducted to observe any possible cleavage

difference in ligand bound and unbound riboswitches. Max and min are the highest and lowest cleavage values for

each altered band in comparison to the control, respectively. The

2.2.1. RNA labeling concentration of TPP required to produce half maximal variation

In order to label the RNAs, 20 pmol of RNA was dephospho- of cleavage provides an estimate of the Kd for TPP–TPP riboswitch

rylated using antarctic phosphatase. After removing the enzyme complex.

by phenol extraction, dephosphorylated RNA was labeled subse-

32

␣

quently with - P-ATP using T4 polynucleotide kinase. RNAs were 2.3. SPR assay

passed through Illustra MicroSpin G-25 Column (GE Healthcare)

and purified over 8% denaturing PAGE (urea 9 M) and eluted from 2.3.1. Immobilization of biotinylated single stranded DNA on SA

gel pieces in crush soak buffer, with subsequent precipitation with chip

◦

ethanol and resuspension in Mili-Q water. All SPR measurements were carried out at 25 C using a Biacore

T100 instrument. DNA immobilization and RNA capturing were

2.2.2. In-line probing done based on the method described by Liu et al. (Liu and Wilson,

 32

In line probing reaction was made containing 1 ␮l 5 P- 2010). Streptavidin coated sensor-chip (SA-chip) were purchased



labeled RNA (20000–100000 cpm), 3 ␮l water, 5 ␮l 2 x In-line from Biacore and a biotinylated single stranded DNA (5 -Biotin-



reaction buffer (100 mM Tris-HCl, 40 mM MgCl2, 200 mM KCl, pH CGTCGCAGATCGTGTCTTCC-3 ) was immobilized on all flow cells.

8.3) and 1 ␮l of TPP solution in the desired concentrations. For HSB-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA,

making a dilution row, different concentrations of TPP were pre- 0.005%, v/v polysorbate 20) was used as running buffer. First, in

pared from 10 mM stock solution in In-line reaction buffer. The order to remove any unbound streptavidine from the chip, activa-

◦

reactions were incubated for approximately 40 h at 25 C. The con- tion buffer (1 M NaCl, 50 mM NaOH) was injected for 1 min (with

trol reactions including RNase T1 RNA digestion (T1) and partial 20 ␮l/min flow rate) seven times. Afterwards, prepared solution

alkaline hydrolysis (–OH) ladders were prepared as 10 ␮l reac- containing 25 nM DNA in HSB-EP buffer was injected with 2 ␮l/min

tions and quenched with 10 ␮l loading buffer. Dried gels were flow rate over the each cell. The injection was stopped once proper

 

Fig. 1. (A) Multiple alignment of 5 UTR of thiCEFSGH of Alishewanella sp. (B) Multiple alignment of 5 UTR of thiC gene in A. tabrizica, E. coli and B. subtilis and

conserved base paired stem-loops (P1, P2, P3, P3a, P4 and P5) are identified. The alignment was carried out using CLUSTAL omega with default parameters.

74

Response Unit (RU) was achieved. The steps were repeated for all ing results. TPP was injected for 2 min at a flow rate of 30 ␮l/min.

of the flow cells. TPP was allowed to dissociate by flushing the cells with running

buffer for 5 min at the same flow rate. Data points were recorded

every second. The binding sensorgrams were obtained from the

2.3.2. Riboswitch loading on SA-chips

subtraction of F2-F1, F3-F1 and F4-F1.

RNA preparation and purification were carried out as previously

described in In-line probing section. The difference was forward

primers which add a complementary region to the biotinylated 2.3.4. Kd determination

single stranded DNA and TTT linker beside to the T7 polymerase The responses were determined from sensorgrams by using

promoter sequence. The solutions of prepared RNA (aptamer BIAevaluation software version 4.0.1. The sensorgrams were fitted

domain of TPP riboswitch) with the concentration of 1 ␮M were with a simplest model for 1:1 interaction between the riboswitch

prepared for injection. Running buffer containing 10 mM HEPES pH aptamer and TPP. Direct curve fitting of sensograms was used to

7.4, 100 mM NaCl, 100 mM KCl, 5 mM MgCl2 was used with starting determine the dissociation equilibrium constant, Kd.

20 ␮l/min flow rate and each type of RNA solution was injected on

each flow cell to get a suitable immobilization amount and flow

3. Results

cell 1 was left without RNA loading. For renewal and changing

the type of riboswitch loading, regeneration buffer (25 mM NaOH)

3.1. Sequence and secondary structure analysis of TPP riboswitch

was injected over flow cells (for 2 min with 10 ␮l/min flow rate)

in A. aestuarii and A. tabrizica

to remove the last RNAs properly. The injection needle was rinsed

with running buffer before injection of new RNA solution.

Among 4 studied microorganisms, only A. tabrizica whole

genome was not available. To get the TPP riboswitch aptamer

2.3.3. Real-time binding experiment for TPP riboswitch-TPP domain from this bacterium, PCR amplification primers were

Binding assays were carried out with a constant flow rate of designed based on the alignment of TPP riboswitches of Alishe-

20 ␮l/min using running buffer containing 10 mM HEPES pH 7.4, wanella species. Having amplified the desired sequence, it was

100 mM NaCl, 100 mM KCl, 5 mM MgCl2. Flow cell 1 immobilized predicted as an aptamer domain of TPP riboswitch in A. tabriz-

with biotinylated DNA was as reference flow cell and other flow ica, using the RegRNA program. Besides, using the same program,

cells were loaded with aptamer domains of TPP riboswitch in A. the sequence of aptamer domains of TPP riboswitches in A. aes-

aestuarii, A. tabrizica, E.coli and B. subtilis. TPP solution was injected tuarii was also identified. The alignment among TPP riboswitches

at least with six different concentrations over all channels. The of Alishewanella species as well as A. tabrizica, E.coli and B. subtilis

range of concentrations was considered between 1 nM − 1 ␮M pre- are shown in Fig. 1A and B, respectively. As shown in the align-

pared in the running buffer by serial dilutions of a 10 ␮M TPP stock ment, the first difference among TPP riboswitches of Alishewanella

solution based on Kd reported in the literature and In-line prob- sp. is their length; while, the aptamer domain is predicted as about

Fig. 2. (A) General secondary structure of TPP riboswitch in Rfam (http://rfam.xfam.org/family/TPP) and secondary structure of TPP riboswitch aptamer domain in (B) A.

tabrizica and (C) A. aestuarii using RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). Conserved stem-loops in the structure are assigned.

75

Fig. 3. (A) In-line probing of 108 nt TPP riboswitch aptamer domain from A. tabrizica. In the right gel, lanes include precursor RNA, T1 digestion and alkaline hydrolysis of

−

RNA (OH ) and RNA incubated without TPP (-TPP) and with TPP (+TPP). In the left gel, different concentrations of TPP (1 mM, 100 ␮M, 10 ␮M, 1 ␮M, 100 nM, 10 nM, 1 nM)

are included. Nucleotides with different intensity between presence and absence of TPP are shown with flags. Conserved structured stem-loops are also assigned in the gel.

(B) the sequence used for In-line probing and secondary structure based on RNAfold prediction with highlighting of flagged nucleotides. (C) The extent of RNA cleavage at

site G69 normalized to G57 intensity is plotted for different concentrations of TPP to estimate concentration of TPP needed to achieve half maximal conformational change

of RNA (apparent Kd).

100 nt in all of them, the expression platform is about 70 nt longer and P4-P5 segments are similar in size and structure. However, the

in A. tabrizica. Furthermore, results showed that the similarity in sequence and secondary structure of the aptamer domain from B.

aptamer domain (starting from position 15 to 112) between A. aes- subtilis has some differences relative to Alishewanella sp. and E. coli.

tuarii, A. jeotgali and A. agri is remarkably high. However, A. tabrizica

contain slightly different nucleotides in positions 28 (C/A), posi-

3.2. In-line probing

tion 35–37 (AT/CGA), 43 (C/G), 47 (A/G),117 (A/G),121 (A/T), 124

(G/A), 126–127 (AT/CC), 128 (C/G), 131 (T/G) which are not co-

To get insights in the secondary structure of the aptamer domain

variations. Comparing expression platform (starting from position

of the TPP riboswitch of A. tabrizica, A. aestuarii and B. subtilis, In-

113 to 370), even with the high difference among the expression

line experiments were conducted. Additionally the apparent Kd of

platform Alishewanella species, there is high similarity in the area

the tested motif against TPP was determined. Figs. 3–5 illustrate

close to start codon (position 311–370). On the other hand the

the In-line probing results of aptamer domains of TPP riboswitch

alignment between TPP riboswitch of A. tabrizica, E. coli and B.

in A. tabrizica, A. aestuarii and B. subtilis, respectively. For A. tabriz-

subtilis (Fig. 1B) showed less similarity even in aptamer domain

ica the most prominent changes in the cleavage pattern due to TPP

sequences. Nevertheless, the identity percentage between E. coli

binding are at nucleotides G92 (P1-P4 junction), A78, U77 (L5), G69

and A. tabrizica in aptamer domain (78%) is more than B. subtilis

(P4), U62 (P2-P4 junction), A43, U39 (P3-P3a junction) (Fig. 3A).

and A. tabrizica (54%) using pairwise alignment which could be the

For the motif of A. aestuarii changes are at nucleotides A79, U78,

result of taxonomical proximity between E. coli and A. tabrizica.

G70, A42, U40 (Fig. 4A). According to structural studies (Serganov

The general secondary structure of the TPP riboswitch extracted

et al., 2006), nucleotides in P4, P4-P5 junction and P2-P4 junction

from Rfam and the predicted structure of the TPP riboswitch

are essentials for pyrophosphate recognition with aiding of a pair

aptamer domain (using RNAfold) from A. tabrizica and A. aestuarii

2+

of hexa-coordinated Mg ions. In addition, nucleotides located

are illustrated in Fig. 2A–C, respectively. Considering the secondary

in P3-P3a junction are necessary for the recognition of 4-amino-

structures of the TPP aptamer domain (Figs. 1B and 2A), conserved

5-hydroxymethylpyrimidine (HMP). Differences in the cleavage

nucleotides form stem P1 and P2, as well as the bridges between P3

pattern upon the addition of TPP for the B. subtilis TPP riboswitch

and P3a, P4 and P5. As shown in Figs. 1B, 2B and 2C, P3 and P3a is

were observed for nucleotides G85, C83, U80, G74, C72, U68, U60,

longer in Alishewanella sp. On the other hand, the thi box or P1-P2

A55, A46, C22 (Fig. 5A). These nucleotides are mostly located in 76

−

Fig. 4. (A) In-line probing of 127 nt TPP riboswitch aptamer domain from A. aestuarii. Lanes include precursor RNA, T1 digestion and Alkalyin hydrolysis of RNA (OH ) and

RNA incubated without TPP (-TPP) and with different concentrations of TPP (1 mM, 100 ␮M, 10 ␮M). Nucleotides with different intensity between presence and absence of

TPP are shown with flags. (B) the sequence used for In-line probing and secondary structure based on RNAfold prediction with highlighting of flagged nucleotides. (C) The

extent of RNA cleavage at site G70 normalized to U51 intensity is plotted for different concentrations of TPP to estimate concentration of TPP needed to achieve half maximal

conformational change of RNA (apparent Kd).

thi box (P4-P5). However, nucleotides located in P2, P3 and P3a 3.3. Surface plasmon resonance spectroscopy

are also involved in binding as explained above. In addition, most

visible intensity changing nucleotides in TPP incubated RNAs were To evaluate the affinity of TPP binding to TPP riboswitch aptamer

determined (Figs. 3A, 4A and 5A) and normalized cleavage fractions domains of different bacteria, equilibrium dissociation constants

were plotted with different concentration of TPP in order to calcu- was measured using surface plasmon resonance (Fig. 6 and Table 1).

late Kd (Figs. 3C, 4C and 5C). For B. subtilis the intensity changes of Overall, the kinetic profile of the interaction between TPP and

nucleotide U80 (Figs. 5A and S1) was employed for Kd calculation. TPP riboswitches demonstrated very fast association and disso-

In A. tabrizica and A. aestuarii the most drastic change is related to ciation rates (Fig. 6). As a result, affinity determination based on

G69 and G70 (P4-P5 junction), respectively. Those were used for the kinetics was impossible to calculate by the instrument. Instead,

calculation of the Kd and for the normalization, the intensity of the affinity determination was carried out by analyzing concentration-

whole corresponding lane was used. Calculated Kd values are pre- dependent steady state responses.

sented in Table 1. Comparing the Kd values of the TPP riboswitches The values of steady state Kd for TPP binding were 4.1 nM,

derived from the studied bacteria (Table 1), the order of TPP affin- 6.8 nM, 46.8 nM, and 548.0 nM for A. tabrizica, A. aestuarii, E. coli,

ity to TPP riboswitch aptamer domain is A. tabrizica > A. aestuarii > B. and B. subtilis, respectively. As a result, the order of TPP affinity to

subtilis. TPP aptamer domains of examined bacteria was shown as A. tabriz-

ica > A. aestuarii > E. coli > B. subtilis which makes the TPP riboswitch

77

Fig. 5. (A) In-line probing of 110 nt TPP riboswitch aptamer domain from B. subtilis. In the gel, lanes include precursor RNA, T1 digestion and alkalyin hydrolysis of RNA

−

(OH ) and RNA incubated without TPP (-TPP) and with 1 ␮M (+TPP) are included. Nucleotides with visible different intensity between presence and absence of TPP are

shown with flags. Conserved structured stem-loops are also assigned in the gel. (B) the sequence used for In-line probing and secondary structure based on Mfold prediction

with highlighting of flagged nucleotides. (C) The extent of RNA cleavage at site U80 normalized to G45 intensity is plotted for different concentrations of TPP to estimate

concentration of TPP needed to achieve half maximal conformational change of RNA (Fig. S1) (apparent Kd).

aptamer domain of A. tabrizica a significantly better aptamer than derivatives (Rodionov et al., 2003), carbohydrates (Winkler et al.,

either of the other tested TPP aptamer domains in terms of binding 2004), nucleobases and their derivatives (Nelson et al., 2015), ions

to TPP. The determined Kds of the SPR measurements are in good (Furukawa et al., 2015) and coenzymes such as thiamine pyrophos-

accordance with those obtained by In-line probing experiments. phate (TPP) (Winkler et al., 2002; Kubodera et al., 2003). TPP as

an active form of thiamine is a cofactor playing an important role

4. Discussion in several enzymatic reactions including glycolysis, the citric acid

cycle and the pentose phosphate pathway. As a result, TPP level

Riboswitches are one of the most important RNA regulatory maintenance is essential for the cell survival. The TPP riboswitch



elements known for their unique characteristics in specific and was one of the first riboswitches determined in the 5 UTR of

selective binding to cellular metabolites such as amino acids and involved in the biosynthesis and transport of thiamine in

78

Fig. 6. Affinity analysis of the TPP riboswitch aptamer from B. subtilis, E. coli, A. tabrizica and A. aestuarii. Plots are the global fit obtained from a 1:1 binding model based on

the experimental data. The apparent equilibrium Kd values are reported in Table 1.

bacteria. Later on, this riboswitch was also discovered in fungi, genome, some primers were designed based on the alignments

algae and higher plants. The thiC gene is involved in the produc- of thiC gene among Alishewanella species. Subsequently, amplified

tion of phosphomethyl pyrimidine synthase enzyme which is the product was sequenced and predicted as TPP riboswitch as well.

first gene in the operon of ThiCEFSGH in E. coli (Vander Horn et al., As shown in Fig. 1A, the aptamer domains of TPP riboswitch in A.

1993) and a separate gene in B. subtilis. This enzyme starts the thi- tabrizica and A. aestuarii (position 15–112) are 97 nt and 98 nt long,

amine biosynthesis pathway with catalyzing the synthesis of the respectively. In addition, despite some slightly differences located

hydroxymethylpyrimidine phosphate (HMP-P) moiety of thiamine in positions 30–50 in A. tabrizica, the aptamer domain is mostly

from aminoimidazole ribotide (AIR) (Lawhorn et al., 2004). The TPP identical in four species of Alishewanella. Nevertheless, consider-



riboswitch located upstream of thiC gene was identified in sev- ing the alignment of 5 UTR of thiC gene among A. tabrizica, E. coli

eral bacteria. However, the only published kinetically studied TPP and B. subtilis (Fig. 1B), the similarity between aptamer domains

riboswitch in the upstream of thiC gene was in E. coli (Winkler et al., is much lower. However, the identity percentage between E. coli

2002). and A. tabrizica is more than the similarity between B. subtilis and

The TPP riboswitch is the only riboswitch identified in eukary- A. tabrizica. This observation is in agreement with the phyloge-



otes so far. For instance, it is located in the 3 UTR of putative thiC netic relation of riboswitches sequences’ conservation (Barrick and

in plants (i.e Poa secunda and Oryza sativa (Wachter et al., 2007), Breaker, 2007; Singh and Sengupta, 2012) as Alishewanella belongs

tomato LethiC gene (Zhao et al., 2011)) and thiC of a green algae C. to the same phylum of E. coli’s (proteobacteria).

reinhardtii (Croft et al., 2007). Considering the mechanism of gene regulation, despite

In the present study, we described the identification of TPP sequence variations in the expression platforms, there is a high sim-



riboswitches located in 5 UTR of thiC operons in A. tabrizica and ilarity (100%) close to start codon (positions 311–370). As position

A. aestuarii, and their comparison with E. coli and B. subtilis TPP 357–360 (GGTG) and 325–328 (CACC) are predicted as Shine Dal-

riboswitches. The thiC operon of A. aestuarii is annotated in NCBI garno sequence (SD) and anti-SD, respectively, this hints for a SD

(accession number: ALAB01000042.1) to code the thiamine biosyn- masking mechanism. However, the first assigned TPP riboswitch

thesis protein ThiC, thiamine monophosphate synthase (thiE like upstream of thiC operon in E. coli was proposed to control gene

protein in E. coli), UBA/THIF-type NAD/FAD binding protein, sulfur regulation by both transcription attenuation and translation termi-

carrier protein ThiS, thiazole synthase (thiG like protein in E. coli) nation (Winkler et al., 2002). Secondary structure prediction results

and thiamine biosynthesis protein ThiH. As a result, the operon is in the same well-known structure for TPP riboswitches including

obviously similar to thiC operon in E. coli. Accordingly, we predicted conserved stem loops P1, P2, P4 and P5 for the newly assigned TPP

the presence of TPP riboswitch in the upstream of this operon in A. riboswitches of A. tabrizica and A. aestuarii. P3 and P3a, as less con-

aestuarii using RegRNA 2.0 (Chang et al., 2013) riboswitch scanning. served parts, are much longer in A. tabrizica and A. aestuarii relative



The predicted 5 UTR of thiC operon in A. aestuarii was synthesized to E. coli and B. subtilis.

in order to study in vitro the TPP binding characteristics of this ele- TPP riboswitch is a type II class as it goes to long distance

ment. However, because of non-availability of A. tabrizica’s whole rearrangements following ligand binding, in contrast to type I 79



Table 1

5 UTRs of thiC genes sourced from A. tabrizica, A. aestuarii, E. coli and

Affinity of TPP binding to TPP riboswitch aptamer domain (Kd) using In-line probing

B. subtilis were compared. In-line probing results of RNAs from A.

and SPR.

tabrizica and A. aestuarii incubating with and without TPP showed

TPP riboswitch aptamer domain Kd (nM) SPR Kd (nM) In-line probing

distinct changes in the cleavage patterns, demonstrating that the

A. tabrizica 4.1 2 studied RNAs are functional TPP riboswitches. Despite the longer

E.coli 46.8 100 (Winkler et al., 2002) P3 and P3a elements in the riboswitch originated from A. tabrizica,

B. subtilis 548.0 120

the structure is similar to the general secondary structure of TPP

A. aestuarii 6.8 26

riboswitches known basically in E. coli. The high degree of similarity



of the 5 UTRs of thiC close to the start codon among Alishewanella

sp. suggests SD masking as the possible mechanism of this type of

riboswitches with local distance rearrangements (Montange and

TPP riboswitch. According to the Kd values determined by In-line

Batey, 2008). Anthoney and colleagues characterized folding and

probing and SPR methods, the affinity of TPP binding was highest in

energy of TPP aptamer originated from Arabidopsis thaliana thiC

A. tabrizica (2 nM determined by In-line probing and 4 nM by SPR)

gene using a single-molecule optical-trapping method. The study

and it was in the order of TPP aptamer domains sourced from A.

confirmed the type II behavior of the riboswitch as well as a hier-

tabrizica > A. aestuarii > E. coli > B. subtilis.

archical folding sequence. Accordingly, secondary structure forms

without the presence of TPP. However, tertiary structure forms

Acknowledgments

slowly and in concert with ligand binding. Moreover, it was pro-

posed that TPP binding progress, from weak to strong binding, may

This article was based on Ph.D thesis of Elnaz Mehdizadeh Agh-

be a final step in the folding process to guarantee selectivity of

dam registered in Tabriz University of Medical Sciences. The Ph.D

riboswitch activation (Anthony et al., 2012).

thesis proposal submitted at No. 80 in the Faculty of Pharmacy,

According to the In-line probing results, TPP riboswitch func-

Tabriz University of Medical Sciences.

tionality of studied sequences was approved. The cleavage sites in

A. tabrizica and A. aestuarii are located in Thi-box (P4 and p5) and

P3-P3a junction which is in accordance with previously studied TPP

riboswitches in bacteria and eukaryotes. However, in B. subtilis, two

cleavage sites following TPP binding (A46 and C22 with reduced

and increased intensity) are located in P3 and P3a, respectively.

According to previous studies, the TPP-bound aptamer adopts a

special folded structure in which one sensor helix arm (P2-P3)

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