L3-4 The Stringent Response
The passive control model?
*Psyn= promotor activity of biosynthesis genes An alternate model suggesting that ppGpp acts indirectly through chnages in RNAP availability rather than directly: This model describes that stringent rRNA promotors require high amounts of RNAP 70 to become saturated but biosynthesis promotes require low levels of RNAP 70 to reach max activity. This shows that activity of promoters and hence ribosome number is dependent on RNAP 70. Ie flexible activity depending on the amount There is a finite amount of RNAP. In rich mediums, RNAP is more active on rRNA operons (as biosynthetic operons are redundant; bac does not need to synthesise own aa as already in medium). Therefore, high avalibelity for rrna operons. In minimal mediums, RNAP focuses activity on biosynthetic operons. Makes sense as if stressed, needs the aa so only needs low amount to be saturated).
What role does pppGpp have in other bacteria?
*growth rate control: pppGpp influences protein synthesis machinery (ie. ribosomes) -> virulence: affects virulence gene expression in Salmonella typhimurium -> biofilm formation: shown to decrease in E.coli relA spoT mutants This shows that the view of ppGpp as primarily a regulator of rRNA transcription has been expanded and it is now clear it has a widespread role in many systems important for cell physiology during growth as well as adjustments to non-growth conditions.
How are rRNA genes regulated?
-> Bacteria can have many copies of rRNA genes (E.coli has 7) this provides flexibility in controlling stable RNA levels. Indeed, mycobacterium tb only has 1; it is slow growing. How growth rate regulates expression...... 1. Strong promotor: Promotor is very string due to being so close to the consensus sequence for RNAP binding. Moreover, a high growth rate means more ATP/GTP available. This favours transcription initiation. 2. UP elements: AT region. Very effective at binding the alpha subunit of RNAP. This increases promotor activity ~50 fold. 3. FIS: Factor for Inversion Stimulation. A high growth rate = high FIS protein levels, which stimulates transcription. 4. H-NS: Histone like Nucleoid restriction protein. A low growth rate = high HNS protein levels, which represses transcription. 5. ppGpp: Interacts with RNAP and represses transcription. However removing FIS and H-NS, the cell can still adjust its level of rRNA in a growth dependent manner----> not the whole story
Enrichment experiments for RelA mutants? What is the significance of these? What is NTG?
-> Important point is that Ampicillin kills actively growing bacteria (inhibits cross linking in peptidoglycan) meaning cells burst under turgor pressure BUT not non-growing bacteria -> Enrichment process: 1. Use auxotrophic bacteria e.g needs leucine to grow 2. Mutate these strains with NTG. Wash to remove the NTG. 3. Resuspend in a medium without leucine. Bacteria STOPS growing. 4. Add leucine and ampicillin. Harvest the cells via centrifuge and repeat. Every run of this cycle leads to a higher % of RelA mutants. How this enriches for RelA mutants: you initially mutate the cells so all cells could be RelA mutants. Then by adding a combination of leucine and ampicillin you are selecting only the cells that do not grow in the presence of added amino acids ie. the RelA mutants,
How does growth rate control ribosome levels?
-> We know that as growth rate increases, ribosome content increases (you need more ribosomes to keeo up with the demand of growth) Growth rate does this by regulating the expression of rRNA genes
What can trigger a stringent response?
1. A shift to a poorer carbon source e.g glucose to succinate 2. Amino acid starvation (very well characterised) (downshift) 3. A shift to a poorer nitrogen source e.g ammonia to nitrate (downshift) 4. Metabolic toxins
How was it discovered that pppGpp interacts with RNAP?
A RelA SpoT double mutant (ie. no pppGpp) was cultured and plated. It is unable to produce ppGpp; this is known as the relaxed response. Without several amino acids (E.coli needs 9) , it will therefore not grow on a minimal medium. -> However, you may see the growth of a single colony. This is a supressor mutant, which has recovered the ability to grow on a minimal medium. -> You can sequence the genome of the original mutant and the suppressor mutant and see how they differ. ie. what about the suppressor mutant reconstitutes its activity? -> Changes in the suppressor mutant were mapped back to the beta/beta' subunits of RNAP. This shows that the mutation facilitating ppGpp mediated effects occurred in RNAP itself.
What is the stringent response?
A global control network- a universal response to stress It is a period of extended lag whilst the bacteria adapts to new conditions •Stop growth + conserve resources •Induce biosynthetic, fuelling & stress survival genes - adapt to new condition
How does SpoT interact with acyl carrier proteins (ACP)?
ACP have a role in fatty acid synthesis; fatty acids are bound to ACP and more can be added to the chain Upon C starvation there will be an increase in short tail fatty acids. These short tail fatty acids interact with SpoT. SpoT has two domains: synthase (ATP + GTP -> pppGpp) and hydrolase (ppGpp -> GDP + Pi) Short tailed ACP will stimulate synthase domain but inhibit hydrolase domain. Therefore, adjusting the synthesis of pppGpp to address perturbations in carbon metabolism
E.coli has a particululary complex stringent response. Describe the more general stringent response that occurs in other bacteria? What are TGS and ACT domains?
E.coli has two main proteins involved in the stringent response (E.coli and SpoT). In the general response there is only one type: RSH (RelA SpoT Homologues) proteins. They are more similar to SpoT than Rel A in that they have a hydrolase, synthase, TGS and ACT domain. Whereas RelA does not have a hydrolase domain. Both synthase and hydrolase domains of RSH have the same function as SpoT (SD: APT + GTP => pppGpp) (HD: ppGPP => GDP + Pi) -> When SpoT* is present synthesis is favoured -> When SpoT is present hydrolysis is favoured This is the case for all bacteria. The only bacteria that do not have a stringent response are obligate intracellular pathogens as they live in a homeostatically controlled constant environment where they are unlikely to require a stress response.
How does pppGpp bring about physiological changes?
Essentially by interacting and binding to RNAP subunits. This is different to most paradigm transcriptional regulators, which in contrast bind to DNA regions in a promotor to affect RNAP. This brings about a whole range of adaptive responses: -> Affect transcription of stable RNA proteins (tRNA, rRNA) -> Translational repression -> rPOS synthesis (involved in general stress response) -> Synthesis of amino acid biosynthesis proteins (figure shows the two parallel SpoT and Rel A pathways that produce ppGpp)
How does ppGpp stimulate transcription of biosynthesis genes? (DIRECT + regulation)
Even less is known than the mechanism of negative regulation but one mechanism proposes that the destabilisation of the open complex actually helps promotor-escape and transcription initiation from these promotors.
Describe how (p)ppGpp was discovered? [central player in the stringent response]
In the 1950s, mutants of the RelA gene of E.coli were shown to continue to make stable RNA in nutrient downshift Through thin layer chromatography, two 'magic spots' were observed. These were nucleotides that accumulated IMMEDIATELY during nutrient downshift. and declines afterwards These nucleotides (I and II)= I: Guanosine tetraphosphate, ppGpp II: Guanosine pentaphosphate, pppGpp They were identified as important signal molecules in the stringent response
Describe the role of the stringent response in bacterial survival during a nutrient downshift ie. amino acid starvation? **is this stringent response or what what happen if sr didnt occur?
In the case of amino acid starvation, the signal trigger the stringent response will be a shortage of CHARGED amino acids. In response to this...... 1. Reduction in protein synthesis 2. Stable RNA synthesis STOPS 3. Ribosomal protein synthesis STOPS 4. Slowing of macromolecular synthesis 5. Adaptive changes occur like the increased synthesis of specific proteins facilitating the biosynthesis of amino acids -> Points 2 and 3 mean that the cell will stop making ribosomes -> Overall stops about 50% of cellular metabolism
How does ppGpp form under the nutrient downshift condition of carbon starvation?
In the case of carbon starvation, the formation of ppGpp doesn't involve a ribosome but is very similar - SpoT is hydrolysed to GDP, which is hydrolysed to GTP then Spot* - SpoT* (stronger synthase domain) different form of SpoT) is hydrolysed to make pppGpp -> Evidence suggests that SpoT can monitor perturbations in carbon metabolism through interactions with acyl carrier proteins
How does DksA bind to RNAP?
Suggested that it protrudes into the secondary channel of RNAP to stabilise the ppGpp-RNAP complex On the basis of structural similarity to GreA (a transcription elongation factor)
How does ppGpp form under the nutrient downshift condition of amino acid starvation? ****************************************************** more details
The ribosomal pathway: - The presence of uncharge tRNA activates the RelA protein - SpoT is hydrolysed to make GDP, which is hydrolyse to make GTP - With the help of the Rel A protein, pppGpp is produced from this
Explain the mechanism of translational repression?
The ribosome assembly: The ribosome has two subunits (30s and 50s) within these subunits are rRNA (16s and 23s). Ribosomal proteins (like S17) can bind to site specific sequences of this rRNA to assemble the ribosome. The stringent response: We know that during the stringent response, transcription of rRNA is inhibited. This leaves these ribosomal proteins with no where to bind. However, S17 can also bind to its own mRNA at the TIR (translation initiation region). In binding to this low affinity binding site, it physically inhibits its own translation.
How does ppGpp inhibit transcription of rRNA genes? (DIRECT - regulation)
These are not mutally exclusive and may be working in concert: 1) Open complex stability ppGpp can destabilise the open complex of all promotors but this may only have negative effects on promotors with intrinsically unstable open complexes 2) Promotor clearance Studies from the λPR promotor show negative effect of ppGpp primarily results from negative effects on promotor clearance (Any process involved in the transition from the initiation to the elongation phases of transcription) 3) Pausing ppGpp increases pausing during transcriptional elongation Other possible mechanisms include base pairing with cytosines, competition between ppGpp and the NTP substrates
What confirmed ppGpp interaction with β and β′ subunits?
co-crystalisation of RNAP structure with ppGpp
How does pppGpp affect transcription and how was this discovered?
pppGpp inhibits the transcription of ribosomal RNA genes but stimulates the transcription of amino acid biosynthesis genes. In this sense, it redirects transcription so that genes important for starvation survival are favoured at the expense of those required for growth. This was shown through a series of in vitro transcription experiments. Moreover, it was shown that DksA protein has a partner role in this. This was eluded to initially as DksA mutants had similar phenotypes as the RelA and SpoT double mutants (ie. unable to grow on minimal medium). In vitro transcription uncovered its role in either further inhibiting (binding to RNAP and decreasing open complex stability) or stimulating transcription (expression of RpoS. (amplifying effects of pppGpp).
