Part:BBa_K3407018

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

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 300
Illegal XbaI site found at 673
Illegal PstI site found at 527
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 300
Illegal PstI site found at 527
21
INCOMPATIBLE WITH RFC[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
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 300
Illegal XbaI site found at 673
Illegal PstI site found at 527
25
INCOMPATIBLE WITH RFC[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
INCOMPATIBLE WITH RFC[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

Expression

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.

References

Ordered List