Difference between revisions of "Part:BBa K1758120"
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<h3>Overview</h3> | <h3>Overview</h3> | ||
| − | The product of the | + | 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. |
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| + | <span class='h3bb'>Sequence and Features</span> | ||
| + | <partinfo>BBa_K1758120 SequenceAndFeatures</partinfo> | ||
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<h3> RraA – Experimental Setup </h3> | <h3> RraA – Experimental Setup </h3> | ||
| − | <p> To characterize RraA <i>in vivo</i>, we created two <i>E. coli</i> strains. One carried the <i>rne</i> gene that codes for RNase E under control of the inducible T7-promoter (P<sub>T7</sub>-<i>rne</i>-plasmid), whereas a second strain carried an additional second plasmid with the RraA coding sequence (P<sub>T7</sub>-<i>rraA</i>-plasmid; <a href="https://parts.igem.org/Part:BBa_K1758122" target="_blank">BBa_K1758122</a>). For <i>in vitro</i> 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. </p> | + | <p> To characterize RraA <i>in vivo</i>, we created two <i>E. coli</i> strains. One carried the <i>rne</i> gene that codes for RNase E under control of the inducible T7-promoter (P<sub>T7</sub>-<i>rne</i>-plasmid), whereas a second strain carried an additional second plasmid with the RraA coding sequence (P<sub>T7</sub>-<i>rraA</i>-plasmid; <a href="https://parts.igem.org/Part:BBa_K1758122" target="_blank">BBa_K1758122</a>). For <i>in vitro</i> characterization, purified RraA that contained an N-terminal 6xHis-Tag (<a href="https://parts.igem.org/Part:BBa_K1758122">BBa_K1758122</a>) was added to our CFPS reaction to see if RraA had the same positive effect in our cell-free setup. </p> |
<h3> RraA – Results </h3> | <h3> RraA – Results </h3> | ||
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<p> The reason for this effect is investigated in detail in <a href="http://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Airen2011">Airen, 2011</a>. 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. </p> | <p> The reason for this effect is investigated in detail in <a href="http://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Airen2011">Airen, 2011</a>. 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. </p> | ||
<p> If one is interested in obtaining high amounts of protein via CFPS, the addition of RraA – or similar positive effectors – is definitely recommended. </p> | <p> If one is interested in obtaining high amounts of protein via CFPS, the addition of RraA – or similar positive effectors – is definitely recommended. </p> | ||
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| + | </br> | ||
| + | <h2>References</h2> | ||
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| + | <p id="Gorna2010"> | ||
| + | Górna, Maria W.; Pietras, Zbigniew; Tsai, Yi-Chun; Callaghan, Anastasia J.; Hernández, Helena; Robinson, Carol V.; Luisi, Ben F. (2010): The regulatory protein RraA modulates RNA-binding and helicase activities of the E. coli RNA degradosome. In RNA (New York, N.Y.) 16 (3), pp. 553–562. DOI: 10.1261/rna.1858010. | ||
| + | </p> | ||
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| + | <p id="Lee2003"> | ||
| + | Lee, Kangseok; Zhan, Xiaoming; Gao, Junjun; Qiu, Ji; Feng, Yanan; Meganathan, R. et al. (2003): RraA: a Protein Inhibitor of RNase E Activity that Globally Modulates RNA Abundance in E. coli. In Cell 114 (5), pp. 623–634. DOI: 10.1016/j.cell.2003.08.003. | ||
| + | </p> | ||
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| + | <p id="Mackie2013"> | ||
| + | Mackie, George A. (2013): RNase E: at the interface of bacterial RNA processing and decay. In: Nature reviews. Microbiology 11 (1), S. 45–57. DOI: 10.1038/nrmicro2930. </p> | ||
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| + | <p id="Yeom2008"> | ||
| + | Yeom, Ji-Hyun; Go, Hayoung; Shin, Eunkyoung; Kim, Hyun-Lee; Han, Seung Hyun; Moore, Christopher J. et al. (2008): Inhibitory effects of RraA and RraB on RNAse E-related enzymes imply conserved functions in the regulated enzymatic cleavage of RNA. In FEMS microbiology letters 285 (1), pp. 10–15. DOI: 10.1111/j.1574-6968.2008.01205.x. | ||
| + | </p> | ||
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</html> | </html> | ||
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Latest revision as of 05:28, 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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 (PT7-rne-plasmid), whereas a second strain carried an additional second plasmid with the RraA coding sequence (PT7-rraA-plasmid; BBa_K1758122). For in vitro characterization, purified RraA that contained an N-terminal 6xHis-Tag (BBa_K1758122) 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, PT7-rne and PT7-rraA. However, induction of T7 polymerase in the strain carrying PT7-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.
References
Górna, Maria W.; Pietras, Zbigniew; Tsai, Yi-Chun; Callaghan, Anastasia J.; Hernández, Helena; Robinson, Carol V.; Luisi, Ben F. (2010): The regulatory protein RraA modulates RNA-binding and helicase activities of the E. coli RNA degradosome. In RNA (New York, N.Y.) 16 (3), pp. 553–562. DOI: 10.1261/rna.1858010.
Lee, Kangseok; Zhan, Xiaoming; Gao, Junjun; Qiu, Ji; Feng, Yanan; Meganathan, R. et al. (2003): RraA: a Protein Inhibitor of RNase E Activity that Globally Modulates RNA Abundance in E. coli. In Cell 114 (5), pp. 623–634. DOI: 10.1016/j.cell.2003.08.003.
Mackie, George A. (2013): RNase E: at the interface of bacterial RNA processing and decay. In: Nature reviews. Microbiology 11 (1), S. 45–57. DOI: 10.1038/nrmicro2930.
Yeom, Ji-Hyun; Go, Hayoung; Shin, Eunkyoung; Kim, Hyun-Lee; Han, Seung Hyun; Moore, Christopher J. et al. (2008): Inhibitory effects of RraA and RraB on RNAse E-related enzymes imply conserved functions in the regulated enzymatic cleavage of RNA. In FEMS microbiology letters 285 (1), pp. 10–15. DOI: 10.1111/j.1574-6968.2008.01205.x.