Part:BBa_K902074

mntP riboswitch

Encodes for the riboswitch used as a regulator in the putative efflux pump mntP. The riboswitch will continue transcription in the presence of manganese and forms the hairpin structure when there is less than 10µM as shown by Waters et al, 2011.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 104
  

Contribution by iGEM Team Tuebingen 2020

Group: Team Tuebingen 2020

Author: Lea Vogt, Lina Widerspick

Summary:

Usage

Riboswitches are small cis-regulatory RNA elements that are co-transcribed with genes subjected to their regulation (reviewed in Serganov and Nudler, 2013 [1]). Binding of their specific target molecule triggers a structural change, that either allows or suppresses the expression of the gene [2]. This feature can be used in synthetic biology applications, where it might be beneficial that riboswitches do not need to be translated [3], unlike regulatory repressors or activator proteins. This putatively allows quick reaction to the presence of a physiological substance. Accordingly, our team, iGEM Tuebingen 2020, decided to use this riboswitch as a core element of our manganese-biosensor. It was combined together with a constitutive Anderson-Promoter (BBa_J23102) or a manganese-inducible promoter (BBa_K902073) to regulate the expression of a fluorescence-tagged (FAST-2-Tag BBa_K3510000) phytochelatin (BBa_K1321005) or a chromoprotein (BBa_K864401). You can find our four different constructs with the following part numbers: BBa_K3510002, BBa_K3510003, Ba_K3510004, and BBa_K3510005.

Biology

In 2011, Waters et. al. mentioned this riboswitch as an “uncharacterized riboswitch regulatory element” when they investigated the transcriptional regulator MntR in E. coli [4]. This is also the paper, where Team Calgary 2012 obtained the sequence for this biobrick. The transcription factor MntR is now known to control manganese homeostasis by repressing the manganese importer MntH [5] and upregulating MntP [4], a putative manganese efflux pump. With some more years of research, Dambach et al. were able to assign the riboswitch to the yybP-ykoY – family [6]. The authors modeled the secondary structure of the first 110 nucleotides of the mntP 5′ UTR, using the yybP-ykoY consensus structure [7] supported by enzymatic and lead structure probing [6]. This part, that corresponds to the yybP-ykoY motif, is highly conserved, while the remainder is more variable [6].

Interestingly, the authors found that the regulation of the MntP manganese exporter consists of two independently contributing elements (Figure 1). First, the inducible promoter (BBa_K902073) allows for transcription, when activated by the regulatory proteins MntR and Fur. These only bind to the promoter region when cells are exposed to high manganese levels and antagonize the repressive effects of histone-like proteins [6]. Second, the riboswitch itself in the 5’ untranslated region (UTR) [6]. Mechanistically, the interaction of manganese with the riboswitch and the thus induced structural change in the element increases the ribosomal binding site accessibility. As a consequence, translation initiation of the co-transcribed gene, MntP, is possible [6].

Figure 1: Regulation of manganese levels in E. coli. During high cellular manganese levels the regulators MntR (yellow) and Fur activate the promoter (green) of the mntP gene (purple). This allows for transcription, but binding of manganese to the riboswitch (orange) is necessary for following translation and expression of the manganese efflux pump MntP (purple). This leads to export of manganese and lowers intracellular manganese concentrations. Additionally, MntR downregulates the expression of MntH (blue) a manganese importer. When little manganese is present in the cell, this downregulation stops and manganese is transported into the cell. At the same time, the riboswitch prevents translation of mntP and manganese export by MntP is decreased.

A 10-min exposure to 10 µM MnCl2 is sufficient for induction of the mntP transcript via this control mechanism [4]. Correspondingly, deletion of mntP or mntR results in heightened sensitivity to manganese and increased intracellular levels. Growth reduction of these phenotypes were specific to manganese, as inhibition could not be reproduced by other metals like zinc, magnesium, iron, nickel, or copper [4].

Further studies have identified the yybP-ykoY riboswitch family functioning as manganese sensor in multiple organisms, namely Escherichia coli [6, 4], Bacillus subtilis [6], Lactococcus lactis [8], Xanthomonas oryzae [9], Streptococcus pneumoniae [10], and it is also suggested for Vibrio cholerae [11]. Suddala et. al. solved the crystal structure of this riboswitch and revealed it forms direct inner-sphere contacts to manganese (II) ion from five phosphoryl oxygen and the N7 of an invariable adenosine [9]. Further, it showed that the manganese binding pocket consists of two distal helical legs docking in a four-way junction (4WJ) [8, 9]. Nevertheless, the structure of the binding pocket does not necessarily seem to be identical for the yybP-ykoY riboswitch from different species [9].

References

1. Serganov A, Nudler E. A decade of riboswitches. Cell 2013; 152(1-2):17–24.
2. Sherwood AV, Henkin TM. Riboswitch-Mediated Gene Regulation: Novel RNA Architectures Dictate Gene Expression Responses. Annu Rev Microbiol 2016; 70:361–74.
3. Garst AD, Edwards AL, Batey RT. Riboswitches: structures and mechanisms. Cold Spring Harb Perspect Biol 2011; 3(6).
4. Waters LS, Sandoval M, Storz G. The Escherichia coli MntR miniregulon includes genes encoding a small protein and an efflux pump required for manganese homeostasis. J Bacteriol 2011; 193(21):5887–97.
5. Patzer SI, Hantke K. Dual repression by Fe(2+)-Fur and Mn(2+)-MntR of the mntH gene, encoding an NRAMP-like Mn(2+) transporter in Escherichia coli. J Bacteriol 2001; 183(16):4806–13.
6. Dambach M, Sandoval M, Updegrove TB, Anantharaman V, Aravind L, Waters LS et al. The ubiquitous yybP-ykoY riboswitch is a manganese-responsive regulatory element. Mol Cell 2015; 57(6):1099–109.
7. Barrick JE, Corbino KA, Winkler WC, Nahvi A, Mandal M, Collins J et al. New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control. Proc Natl Acad Sci U S A 2004; 101(17):6421–6.
8. Price IR, Gaballa A, Ding F, Helmann JD, Ke A. Mn(2+)-sensing mechanisms of yybP-ykoY orphan riboswitches. Mol Cell 2015; 57(6):1110–23.
9. Suddala KC, Price IR, Dandpat SS, Janeček M, Kührová P, Šponer J et al. Local-to-global signal transduction at the core of a Mn2+ sensing riboswitch. Nat Commun 2019; 10(1):4304.
10. Martin JE, Le MT, Bhattarai N, Capdevila DA, Shen J, Winkler ME et al. A Mn-sensing riboswitch activates expression of a Mn2+/Ca2+ ATPase transporter in Streptococcus. Nucleic Acids Res 2019; 47(13):6885–99.
11. Fisher CR, Wyckoff EE, Peng ED, Payne SM. Identification and Characterization of a Putative Manganese Export Protein in Vibrio cholerae. J Bacteriol 2016; 198(20):2810–7.

Mathematical Modeling by iGEM BostonU 2024

Group: BostonU 2024

Author: Kevin Li

Summary

We determined a mathematical model for a linear relationship between manganese concentration and the activity of manganese-inducible genetic parts (encompassing this mntP riboswitch).

Purpose

We designed a device that could quantify the amount of manganese in liquid samples. In order to be able to create biosensors capable of manganese detection, we introduced manganese-inducible genetic parts into E. coli. Specifically, we used the mntR protein (BBa_K902030), which interacts with the mntP promoter (BBa_K4217000) to regulate the activity of the mntP riboswitch (BBa_K902074, this part). In this system, transcription downstream of the mntP riboswitch is activated in the presence of manganese ions. In order to specifically quantify manganese concentrations, we measured changes in the activity of the genetic parts (through measurable fluorescent outputs) at different manganese concentrations. We included a YFP coding region after the coding region of the mntR protein and a GFP coding region after the mntP riboswitch. Thus, YFP expression will represent the activity of the mntR protein and GFP expression will represent the activity of the mntP promoter and riboswitch.

Results

We determined a model for a linear relationship between power-transformed manganese concentration values and change in GFP expression divided by change in YFP expression. The equation for this linear relationship is shown below. Initial (i) measurements were taken immediately after our biosensors were introduced to manganese liquid solutions and final (f) measurements were taken 8.5 hours later. Non-transformed manganese concentration values can be recovered by raising the output of the model to the power of the reciprocal of 0.225. We recommend using our model to accurately detect manganese concentrations in the range of 10 μM to 10 nM. The data used to trained the model as well as the shape of the model is shown in Figure 1.

Figure 1: Linear model for power-transformed manganese concentration against change in GFP expression divided by change in YFP expression overlaid over original data. The slope of the curve is approximately 0.934 and the y-intercept is approximately -0.96. All fluorescence measurements were taken in RFUs (relative fluorescence units).

Methods

Data was collected through heavy metal assays. An equation for a linear model was determined from the data through ordinary least squares linear regression. More information can be found on our wiki on our Contribution, Model, and Protocol pages. Our home page is linked here.