Difference between revisions of "Part:BBa K1758120"

Revision as of 00:59, 19 September 2015

RNase E regulating protein RraA

Overview

The product of the RraA-gene has been reported to interact with RNase E from E. coli and to alternate its activity. Airen showed that if RraA-protein is added to a cell free protein synthesis reaction, the productivity raises about 30% (Airen, 2011). He postulated that activity of RNase E in the reaction is lowered due to the interaction with RraA-protein, therefore mRNA-levels are stabilized. RNase E is brought to the in vitro reaction by E. coli cell extract itself.

Positive Effector RraA – Usage and Biology

The product of the rraA gene (Regulator of ribonuclease activity A, former menG) has been reported to interact with RNase E from E. coli and to alternate its activity (Lee et al. 2003,Yeom et al. 2008, Gorna et al. 2010). RNase E is coded by the rne gene and is essential for E. coli. The protein takes a dual role in the bacterium as it enables processing of important RNAs but also participates in nonspecific degradation of RNA (Mackie 2013).

Airen showed that if RraA-protein is added to a cell-free protein synthesis reaction, the productivity raises about 30% in his cell-free system (Airen, 2011). He postulated that activity of RNase E in the reaction is lowered due to the interaction with RraA-protein, therefore mRNA-levels are stabilized.

RraA – Experimental Setup

To characterize RraA in vivo, we created two E. coli strains. One carried the rne gene that codes for RNase E under control of the inducible T7-promoter (P_T7-rne-plasmid), whereas a second strain carried an additional second plasmid with the RraA coding sequence (P_T7-rraA-plasmid; BBa_K1758122). For in vitro characterization, purified RraA that contained an N-terminal 6xHis-Tag was added to our CFPS reaction to see if RraA had the same positive effect in our cell-free setup.

RraA – Results

In the in vivo experiment there was no observable growth drop when T7 polymerase was induced in the strain carrying both plasmids, P_T7-rne and P_T7-rraA. However, induction of T7 polymerase in the strain carrying P_T7-rne-plasmid only lead to a clear growth inhibition. This difference was apparent although the strain carrying rne-plasmid altogether grew slower than the double transformed strain. Therefore we conclude RraA overexpression rescues E. coli by decreasing activity of RNase E.

Growth curves of E. coli expressing RNase E (rne) and RraA. Two cultures were induced to express T7 polymerase at 1.25 h.

To verify the effect in vitro, RraA in 50 mM HEPES buffer at pH = 7.2 was added to CFPS reactions to a final concentration of 0.3 mg/mL. To exclude that an observed effect resulted from the buffer alone, two control reactions were additionally performed. In these reactions RraA was omitted and the missing volume was filled up with water and buffer only, respectively.

RraA improves final fluorescence signal when added to the reaction. Final concentration of RraA in the reaction was 0.3 mg/mL.

Purified RraA after SDS-PAGE. Protein identity was proven via mass spectrometry.

We could verify Airens observation that RraA is a positive effector in cell-free protein synthesis. More precisely, the results of Airens and our experiment are similar: In our reaction the final signal raised about 33.8 ± 4.5 %, and in Airens experiment the signal raised 28.6 ± 3.1 % respectively when compared to a reaction were RraA is not present (Airen, 2011).

The reason for this effect is investigated in detail in Airen, 2011. The shape of the fluorescence signal curve when RraA is present (green dots) indicates that the protein acts as stabilisator in our reaction. It is likely that by reducing RNase E activity, the rate of mRNA and rRNA degradation is slower.

If one is interested in obtaining high amounts of protein via CFPS, the addition of RraA – or similar positive effectors – is definitely recommended.

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
  COMPATIBLE WITH RFC[1000]