User:Ramasamy K/sandbox

For the Stringent Response stub see [| Stringent response]



[| Stringent Response] is a metabolic regulation pathway.

Introduction

 * Bacteria adapt to environmental stress by selectively expressing certain genes and down regulating genes whose function is not important. This is to reduce the unnecessary use of nutrients. They may come across stress like antibiotics, scarcity of nutrients, temperature (heat/cold shock).  During such times they change their growth rate, change the expression of genes, form spores etc. In many cases few proteins are affected by the external stimuli directly and they in turn bring about the change in the organism.  The stringent response occurs in response to scarcity in amino acids, fatty acids, heat shock etc. In case of amino acid and fatty acid scarcity ppGpp was produced in many bacteria. This small molecule then drastically changes the gene expression of the organism.

History

 * In 1952 scientists reported that the mRNA amount was reduced in response to amino acid starvation. Later genetic studies found that relA was related to this effect, as mutants in relA didn’t respond to amino acid starvation. In 1969 it was found that ppGpp and pppGpp were key molecules involved in the process. Later research found that (p)ppGpp synthesis and breakdown was done by proteins rsh (Rel Sto homolog). In 2000 it was found that ppGpp was also involved in signalling in A. thaliana. In A.thaliana it was found to interact with its host defense proteins. Stringent factors were also found to be involved in pathogenesis. Thus they provide a target for new drug.

Synthesis and breakdown of ppGpp

 * ppGpp and pppGpp are synthesised by phosphorylation of GDP and GTP respectively. ppGpp is also produced by dephosphorylation of pppGpp. [1] Under normal conditions (p)ppGpp levels are maintained at steady level. In E.coli it was found that RelA synthesised (p)ppGpp and SpoT degraded it. But SpoT was also found to have synthase activity. It was found later that homologs of the above proteins have both synthase and hydrolase domains. The namesakes of RSH are the RelA and SpoT proteins of E. coli; two apparently similar RSH proteins exist among many bacteria. Some bacteria have a single RSH protein which has both hydrolase and synthase activity.
 * The active site of RSH is present in the N-terminal side. The RelA has both hydrolase and synthase domains but its hydrolase domain is not active. As a result it has only synthase activity. SpoT is specialised for hydrolysis activity but it also has a weak synthase activity. They require Mn+2 ions for their hydrolase activity.[2]

Regulation of ppGpp level

 * Reciprocal regulation of hydrolase and synthase is required to prevent a futile cycle. During the scarcity of amino acids an uncharged tRNA binds to the A site of the ribosome. This causes a conformational change in the Carboxy terminal Domain (CTD). This change in CTD activates the synthase and pppGpp is produced from GTP and ATP. The terminal phosphate can then be cleaved to produce ppGpp. CTD is essential for this activity. This shows that the signal for synthesis of ppGpp passes from Ribosome to NTD through CTD.
 * Fatty acid scarcity also affects ppGpp level. When there is lesser amount of fatty acid the ratio of free ACP (Acyl Carrier Protein) to fatty acid bound ACP is high, as a result free ACP binds to SpoT in its CTD. In this case the weak synthase activity of SpoT is activated and the hydrolase is inactive. The SpoT then produces ppGpp which then activates relA.

Regulation of transcription by ppGpp

 * A basal level of (p)ppGpp is required for synthesis of certain amino acids. If ppGpp is completely removed the cells don’t grow without amino acid supplement. High level of ppGpp caused decrease in mRNA level.
 * ppGpp interacts with ββ'σ70 subunits of the RNA polymerase and changes its specificity. It downregulates the synthesis of rRNA. As a result ribosomal protein synthesis is also down regulated. This reduces the amount of ribosome present in the cell and thus the rate of transcription.
 * There is an increase in expression of genes required for amino acid synthesis, stationary phase genes etc. ppGpp induces expression of certain genes for amino acid synthesis. The activation can be either direct or indirect. In direct activation the ppGpp directly binds to the polymerase and changes the specificity. In indirect activation the RNAP disintegrates from the promoters and it becomes available for weak σ subunits, which usually don’t bind in low concentration of the RNAP.

Regulation of Growth by ppGpp

 * The rate of growth is decreased by ppGpp due the decrease in rate of transcription. It inhibits DNA replication by interfering with the replication machinery. It was found to inhibit DNA primase.

Conclusion:

 * ppGpp like cAMP is a important regulatory molecule which changes the transcription of a large fraction of genes. It senses the starvation state and induces sporulation. Also it imparts virulence to the bacteria. In M.tuberculosis strain mutant in RelA didn’t survive long time compared to wild type.3 ppGpp null mutants have less pathogenecity . Using ppGpp null mutants for vaccination is being studied. ppGpp is also found in higher eukaryotes. The remnants of stringent pathways in plants show the evolutionary relationship.