Designed by: Javier Navarro Delgado   Group: iGEM20_TUDelft   (2020-10-23)

Mini-3 endoribonuclease with araBAD promoter - RBS - and rrnBT1 terminator

Sequence and Features

Assembly Compatibility:
  • 10
    Illegal EcoRI site found at 300
    Illegal XbaI site found at 673
    Illegal PstI site found at 527
  • 12
    Illegal EcoRI site found at 300
    Illegal PstI site found at 527
  • 21
    Illegal EcoRI site found at 300
    Illegal BglII site found at 309
    Illegal BamHI site found at 809
    Illegal XhoI site found at 818
  • 23
    Illegal EcoRI site found at 300
    Illegal XbaI site found at 673
    Illegal PstI site found at 527
  • 25
    Illegal EcoRI site found at 300
    Illegal XbaI site found at 673
    Illegal PstI site found at 527
    Illegal AgeI site found at 74
  • 1000
    Illegal SapI site found at 56

Usage and Biology

This part was used to Express mini-3 from B. subtilis (BBa_K3407002)

The CDS encodes for mini-3, a small enzyme of 17,9 kDa (when his-tagged) involved in 23S and 50S rRNA processing, coded in the mrnC gene of Bacillus subtilis (UniProt ID:O31418). It belongs to the RNaseIII superfamily of nucleases of double stranded RNA (dsRNA) where enzymes are classified based on the presence of various functional elements associated to the catalytic domain [1]. Although Mini-3 shares high sequence homology with RNAseIII catalytic domains, it does not possess the usual double-stranded RNA binding domain (DRBD) nor any other accessory one, which led to the creation of a new class of RNAseIII (class 4) to include it [1][2].

  • Figure 1: Cleavage of double-stranded RNA (dsRNA) by Mini-3

Unlike other classes of RNAseIII showing secondary structures preferences in cleavage [3], mini3 shows a high sequence associated cleavage, cutting dsRNA containing “ACCU” with little off-targets [4], leaving 3’ protruding overhangs (Figure 1). Behaving as a counterpart of the known DNA restriction enzymes, these dsRNA sequence-dependent endoribonucleases are notably scarce at the moment in protein databases.

  • Figure 2: Crystal structure of mini-3 dimer. The structure has been resolved by [4] available in PDB with 4OUN accession number.

Due to the clear interest in RNA manipulation, the European Commission supports the development or discovery of new dsRNA endoribonucleases of these characteristics [5]. Despite its high sequence similarity to RNAseIII, its interaction with dsRNA differs as the predicted contact is performed by dimers with its major grooves (Figure 2) [4]. It represents a valuable tool to accurately process synthetically designed RNA-based systems. Its crystal structure has been resolved (PDB cod: 4OUN) and RNA binding models have been performed [4], showing a proximal N and C- terminals located at the opposite side of the catalytic site, opening the possibilities to create fusion proteins without hampering RNA catalysis.

Experimental results


To overexpress Mini-3, we incubated E. coli BL21 (DE3) (negative control) and E. coli BL21 (DE3) transformed with the plasmids pBbE8c_mini3. When the cultures reached OD600 ≈ 0.6, they were induced with 20 mM L-arabinose for 4 hours at 37ºC and overnight at 20ºC. The total protein content of the cells was analysed by SDS-PAGE electrophoresis (Figure 3).

  • Figure 3: SDS-PAGE gel showing the overexpression of mini3. E. coliBL21 (DE3) is the negative control and E. coli BL21 (DE3) pBbE8c_mini3 contains the part BBa_K3407018. MW (Molecular weight marker, #1610363 Bio-Rad), PI (pre-induction), 4h (4 hours after induction), ON (overnight). All the samples used corresponded to the same OD600.

As seen in the SDS-PAGE of the total protein content (Figure 3), a band corresponding to the molecular weight of the C-terminal His-tagged Mini-3 shows that it was successfully overexpressed after induction with 20 mM L-arabinose. No additional bands can be observed in E. coli BL21 (DE3) pBbE8c_mini3 without induction, indicating that there is no leaky expression.


Ordered List

  1. Olmedo, G., & Guzmán, P. (2008). Mini-III, a fourth class of RNase III catalyses maturation of the Bacillus subtilis 23S ribosomal RNA. Molecular Microbiology.
  2. Redko, Y., Bechhofer, D. H., & Condon, C. (2008). Mini-III, an unusual member of the RNase III family of enzymes, catalyses 23S ribosomal RNA maturation in B. subtilis. Molecular Microbiology.
  3. Nicholson, A. W. (2014). Ribonuclease III mechanisms of double-stranded RNA cleavage. Wiley Interdisciplinary Reviews: RNA.
  4. Głów, D., Pianka, D., Sulej, A. A., Kozłowski, Ł. P., Czarnecka, J., Chojnowski, G., … Bujnicki, J. M. (2015). Sequence-specific cleavage of dsRNA by Mini-III RNase. Nucleic Acids Research.
  5. Klincewicz, K. (n.d.). Stairway to Excellence Cohesion Policy and the Synergies with the Research and Innovation Funds Example of Synergies Molecular scissors for double stranded RNA-International Institute of Molecular and Cell Biology (IIMCB).