Part:BBa_K1349001
relA
The relA gene encodes a ppGpp synthetase. In bacteria, ppGpp (guanosine 3'-diphosphate 5-' diphosphate) acts as a signaling molecule that regulates a variety of cellular metabolisms in response to changes in the nutritional state of the cells.
Validation of the RelA part
The relA gene encodes a ppGpp synthetase. In bacteria, ppGpp (guanosine 3'-diphosphate 5-' diphosphate) acts as a signaling molecule that regulates a variety of cellular metabolisms in response to changes in the nutritional state of the cells.
This part was designed to allow the rapid synthesis of the alarmone ppGpp, responsible for the stringent response in E. coli. In the context of our project ppGpp is accumulation used to stop initiation of cell division.
How we tested the BBa_K1349001 part:
Expression of the BBa_K1349001 part is expected to cause accumulation of ppGpp and so reduce growth rate of a wild-type cell. At the opposite, expression of this part in a MG1655∆relA mutant unable to make ppGpp is expected to complement the mutant.
As seen on the figure below, our part is functional as it is able to fully complement the MG1655∆relA mutant. See lab-book for details on the experimental procedure.
Attribution of the figure: Dr. Emmanuelle Bouveret.
Conclusion
The protein encoded by out relA part is able to complement a RelA mutant thus demonstrating the functionality of our part.
Improvements of part BBa_K639001
Our RelA brick (BBa_K1349001) represents a significant improvement on the previously available RelA brick made by the 2011 Trondheim team (Part:BBa_K639001). In the first place our brick has no amino acid mutations and no upstream unwanted insert. Secondly our brick lasks the PstI site and thus is compatible with the assembly standards RFC10, 12, 21, 23, 25 and 1000.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1708
Contribution
Group: Qdai 2020
Author: Jiaxin Wu
When Escherichia coli cells are exposed to extreme starvation, synthesis of their rRNA is quickly curtailed for adapting the undernourished environment. A phenomenon called stringent response is known as growth rate-dependent regulation, along with upstream activation and antitermination, including at least four different examined rRNA expression which bear on those changing environments in rRNA synthetic capacity. The stringent response is a description of the elaborate set of adjustment that the cells makes in response to starvation for amino acids, and two hallmarks of the response have been defined, which are identified as guanosine tetraphosphate(ppGpp) and guanosine pentaphosphate (pppGpp). Another significant characteristic of this response has been conducted, which is the reduction of the elongation rate of RNAP molecules. When cells are tripped in an condition lacking the essential amino acid, the normally low level of (p)ppGpp shows a dramatically increase in a few seconds after starvation commences, reach a maximum between 5 and 15 min later, and then decrease to a new steady stage which is 10- to 20-fold higher than the original basal level.
Escherichia coli cells which enter a phase of starvation for Pi induce accumulation of ppGpp, which is associated with relA and depends on the spoT gene product. The synthesis of (p)ppGpp during amino acid starvation is related with relA protein, which is located at 60 min on the E.coli chromosome and encodes the (p)ppGpp synthetase I enzyme. When there are insufficient amounts of a particular charged tRNA in the cell to satisfy the requirement for protein synthesis, the synthesis reaction starts, transferring pyrophosphate from ATP to DTP or GDP to yield pppGpp or ppGpp, respectively. When it comes to overexpress the relA gene with sufficient amino acids in the meantime, the more ppGpp is producted. The first observed effect of the stringent response was the inhibition of stable RNA synthesis, which was attributed to the accumulation of ppGpp, however attempts to measure the effect have not been conducted.
It is also confirmed that PolyP accumulation in Escherichia coli cells depends on an insufficient supply of acids in a Pi-limited medium. According to the results of in vivo experiments, after increasing the concentration of amino acids in the Pi-limited, the accumulation of polyp in the wild-type cells was almost completely abolished(Fig. 1B), and cells growing in the Pi-limited medium without amino acid supplementation accumulated polyp up to a maximum of 8 nmol/mg of protein(Fig. 1C), whereas the cells growing in high-Pi medium(2mM) accumulated only about 1 nmol/mg of the protein(Fig. 1D).
When it comes to the cor_rela_tion among PPX, ppGpp and pppGpp, influence of pppGpp and ppGpp on activitity of PPX-Accumulation of polyP by E. coli shows the removal of polyP can be attributed to the presence or absence of PPX, the principal polyphosphate of E. coli. The effects of these compounds on PPX activity were strikingly inhibitory at 100 μM, pppGpp inhibited PPX by 90%, ppGpp also inhibited PPX, but less strongly(Fig.2).
Another significant connection among the three componuds is PPX possesses pppGpp Hydrolase Activity, and pppGpp is hydrolyzed to ppGpp during this reaction. The principal known route is the action of GppA, which also proved to have exopolyPase activity. In view of the considerable sequence homology between GppA and PPX, the latter was examined and also proved to have pppGpp hydrolase activity (Fig. 3).
Kinetic Features of Inhibition of PPX by pppGpp and ppGpp – Inhibition of PPX, examined at several levels of pppGpp, was consistent with its behavior as competitive inhibitor of polyp substrate with a Ki value of 10 μM (Fig.4).
Stimulation of polyp accumulation by ppGpp and pppGpp could be demonstrated in the course of polyP synthesis by PPK and hydrolysis by PPX (Fig. 5).
Fig.1 PolyP accumulation depends on amino acid (aa) concentration in the growth medium
Fig.2 Inhibition of PPX by pppGpp and ppGpp
Fig. 3 Hydrolysis of pppGpp to ppGpp by PPX
Fig. 4 Lineweaver-Burk plot for inhibition of PPX by pppGpp
Fig. 5 Stimulation of polyp accumulation by pppGpp and ppGpp in the course of PolyP synthesis by PPK and hydrolysis by PPX
References
RAO, N. LIU, S. KORNBERG, A. 1998. Inorganic Polyphosphate in Escherichia coli: the Phosphate Regulon and the Stringent Response. JOURNAL OF BIOLOGY. 180(8). PP. 2186-2193.
Kuroda, A. Murphy, H. Cashel, M. Kornberg, A. 1997.Guanosine Tetra – and Pentaphosphate Promote Accumulation of Inorganic Polyphosphate in Escherichia coli. JOURNAL OF BIOLOGICAL CHEMISTRY. 272(34). PP. 21420-21243.
CONDON, C. SQUIRES, C. SQUIRES, C.L. 1995.Control of rRNA Transcription in Escherichia coli. MICROBIOLOGICAL REVIEWS. 59(4). PP. 623-628.
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