Adaptation to any undesirable change in the environment dictates the survivability of many microorganisms. Such changes generate a quick and suitable response, which guides the physiology of bacteria. Stringent response is one of the mechanisms that can be called a survival strategy under nutritional starvation in bacteria and was first observed in E. coli upon amino acid starvation, when bacteria demonstrated an immediate downshift in the rRNA and tRNA levels (Stent and Brenner 1961). Mutations that rendered bacteria insensitive to amino acid levels were mapped to an ‘RC gene locus’, later termed relA because of the relAxed behavior of the bacteria (Alfoldi et al. 1962). Later on, Cashel and Gallant, showed that two “magic spots” (MSI and MSII) were specifically observed in starved cells when a labeled nucleotide extract of these cells was separated by thin layer chromatography (Cashel and Gallant 1969). These molecules were found to be polyphosphate derivatives of guanosine, ppGpp and pppGpp (Cashel and Kalbacher 1970; Sy and Lipmann 1973), and were shown to be involved in regulating the gene expression in
the bacterial cell, demonstrating a global response, thus fine-tuning the physiology of
the bacterium. Two proteins in E. coli, RelA and SpoT, carry out the synthesis and
hydrolysis of these molecules, respectively, and maintain their levels in the cell
(Cashel et al. 1996; Chatterji and Ojha 2001). On the other hand, Gram-positive
organisms have only one protein Rel carrying out the functions of both RelA and
SpoT (Mechold et al. 1996; Martinez-Costa et al. 1998; Avarbock et al. 1999).
Although Rel or RelA/SpoT has been studied from several systems in detail pertaining to the physiological adaptation, less information is available on the egulation of the protein activity under different conditions. Our studies show that the
RelMsm is composed of several domains (HD, RSD, TGS and ACT) with distinct function. HD and RSD domains, present in the N-terminal half of the protein, harbor catalytic sites for the hydrolysis and the synthesis of (p)ppGpp, respectively. TGS and ACT domains, on the other hand, are present at the C-erminal half of the protein and have regulatory function. It, therefore, appears that a communication exists between these domains, to regulate protein activity. It was shown earlier, while studying Rel from S.equisimilis, that there exists an interaction between the C-terminal and the N-
terminal of the protein which determines the kind of activity (synthesis/hydrolysis),
the protein should demonstrate (Mechold et al. 2002). Later, the N-terminal half
crystal structure of the same protein suggested an inter-domain “cross-talk” between the HD and the RSD domain that controls the synthesis/hydrolysis switch depending on cellular conditions (Hogg et al. 2004).
In the present work, studies have been carried out to understand a Gram-
positive Rel in greater detail and to find out how the opposing activities of Rel are
regulated so that a futile cycle of synthesis and hydrolysis of (p)ppGpp, at the expense of ATP, can be avoided. The work has been divided into several chapters describing
studies on various aspects of the protein.
Chapter 1 outlines the history of the stringent response and summarizes the
information available about the stringent response in various systems including plants.
Several roles that (p)ppGpp plays in different bacteria have been examined. A special mention on the crystal structure of RelSeq has been made with respect to the regulation of activity. Also, the information available regarding the effects of (p)ppGpp on RNA polymerase has been documented. Role of ppGpp in plants has been discussed in great detail with special emphasis on abiotic stresses.
Since different functional domains have been identified in RelMsm, the protein
has been divided into two halves and they have been discussed separately in the form
of two chapters.
Chapter 2 describes the N-terminal half of the Rel protein of M. smegmatis in greater detail. Out of the several domains identified, the role of the two domains
present in the N-terminal half of the protein has been studied. The N-terminal half
shows both synthesis and hydrolysis activities. Importantly, we find that the protein is active even in the absence of accessory factors such as ribosome and uncharged tRNA, unlike RelA of E. coli. Moreover, deletion of the C-terminal half of the protein leads to a much higher synthetic activity, clearly indicating that the C-terminus is involved in regulating the activity of the protein. Both TGS and ACT domains (the two domains found in the C-terminal half of the protein) have been found to play a regulatory role. The results also indicate that all the deleted constructs are active both in vitro and in vivo.
Chapter 3 discusses the C-terminal half of the protein and its role in the
multimerization observed in RelMsm. We show that multimerization of Rel protein is
due to the inter-molecular disulfide cross-linking. Furthermore, we find that the
monomer is the active species in vivo. One of the fascinating points about the C-
terminal half is that it is largely unstructured. Additionally, the C-terminal half cannot complement the N-terminal part of the protein when provided in trans, demonstrating further, the requirement of an intact protein for bringing about regulation of Rel activity. This requirement in cis suggests the presence of an intra-molecular
communication between the N- and the C-termini, as a mediator of protein regulation.
Further, presence of uncharged tRNA increases pppGpp synthesis and down-regulates
its hydrolysis in the wildtype protein. However, the uncharged tRNA-mediated
regulation is absent in the deleted construct with only the N-terminus half, indicating that uncharged tRNA binds to the C-terminal half of the protein. Several cysteine mutants have been constructed to understand their role in the regulation of Rel activity. The results suggest that one cysteine, present at the C-terminus, is required for intra-molecular cross-talk and the uncharged tRNA-mediated regulation.
A detailed characterization of the communication between the two halves of
the protein has been attempted in Chapter 4. Surface plasmon resonance experiments
carried out on the different cysteine mutants discussed in Chapter 3, for uncharged
tRNA binding indicate that all the mutants bind to uncharged tRNA with near-equal
affinities as the wildtype protein. This study suggests that the non-responsiveness for tRNA seen in one of the cysteine mutants is due to the loss of inter-domain
interaction, while the binding of protein to accessory factors is unaffected. Fluorescence resonance energy transfer has been carried out to observe domain
movement in the presence of accessory factors. Distances between the different
domains scattered in this ~90 kDa protein, measured by FRET technique, are suggestive of an inter-domain cross-talk, specifically between C338 and C692, thereby regulating the activity of this enzyme. We show, for the first time, that the product of this protein, (p)ppGpp can bind to the C-terminal half making it unstructured, and can, therefore, regulate the protein activity.
Chapter 5 is an effort to characterize the promoter of rel from M. tuberculosis. This study was undertaken in order to develop an expression system in mycobacteria. The +1 transcription and the translation start sites have been identified. The –10
hexamer for the RNA polymerase binding has also been mapped using site-directed
mutagenesis and is found to be TATCCT. This promoter is also unusually close to the +1 transcription start site. The promoter is specific for mycobacteria and does not
function in E. coli. Additionally, the promoter is found to be constitutive in M.
smegmatis; however, the possibility of it being regulated in M. tuberculosis cannot be
ruled out.
Appendix section discusses, in short, the phylogenetic analysis of the mycobacterial Rel sequences. Diagrams of the plasmids used in this study have been provided. Mass spectra recorded for the in vitro synthesized and purified pppGpp and
the trypsin digest of the full-length Rel protein have also been given.
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| Identifer | oai:union.ndltd.org:IISc/oai:etd.ncsi.iisc.ernet.in:2005/330 |
| Date | 07 1900 |
| Creators | Jain, Vikas |
| Contributors | Chatterji, Dipankar |
| Source Sets | India Institute of Science |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Rights | I grant Indian Institute of Science the right to archive and to make available my thesis or dissertation in whole or in part in all forms of media, now hereafter known. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. |
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