The Stringent Response Flashcards
Bacterial metabolism is regulated by
small molecules
- secondary messengers: ppGpp + cyclic di-GMP
- quorum sensing molecules (AHLs)
secondary messengers are
molecules produced in response to a stimulus and then detected by something else to mediate a response
The stringent response causes a
global decrease in rna synthesis
ppGpp is a normal guanine diphosphate with a phosphate attached on the 3’ end
particularly affects rRNA and tRNA synthesis
by reducing the amount of tRNA and rRNA the cell will reduce the rate of translation which will conserve the a.a. supply
ppGpp is an (3)
ALARMONE
alarm signal that signals the need to shut down RNA production
mediates the stringent response
it is an effector that can be phosphorylated to pppGpp
how do cells sense a shortage of an amino acid
ribosomes stall on mRNA at codons with uncharged tRNAs as they enter A site (with no a.a. on them due to shortage)
this activates RelA
pppGpp is made by
RelA using pppGpp synthase
pppGpp converted back to ppGpp by pppGpp phosphohydrolase
RelA is an enzyme on
approx 1 in 200 ribosomes
RelA is activated when an uncharged tRNA enters the A site of the ribosome
SpoT can also
make and get rid of pppGpp
SpoT also responds to C, P and Fe starvation and oxidative stress
pppGpp binds to
RNA polymerase
stops transcription of rRNA and tRNA, ribosomal proteins, cell wall production and DNA replication
pppGpp turns on
stress proteins
amino acid biosythesis pathways
recycling proteolases
stationary phase sigma factors
What is particularly sensitive to pppGpp in rRNA and tRNA?
the discriminator region
this is a promotor with a high GC content in the -10 to +1 region
this region melts during transcription initiation and the high GC content makes this difficult
promotors which have a high GC content are more sensitive to pppGpp
How does ppGpp work?
makes open complex formation allosterically unfavourable, especially for GC rich discriminators
this is because it binds to RNA pol and affects the ability of the transcription bubble to form
AT rich sequences are less affected
AT rich sequences can be
activated by pppGpp
as transcription slows, more rRNA in the cell = more AT rich sequences transcribed = more stringent response proteins
RNA pol is a major transducer of
pppGpp signal
Outside reading info
secondary effects of ppGpp’s role in regulating expression of other gene products or binding to enzymes other than RNAP. It has been reported that ppGpp affects the activities of DNA primase, lysine decarboxylase, IF2, and guanylate kinase, regulating processes as varied as DNA replication, translation, and
central metabolism. Furthermore, ppGpp is required for virulence in many pathogens
ppGpp’s effects are amplified by DksA, a 17.5 kDa protein that modifies RNAP by binding to the enzyme’s secondary channel
RelA-dependent (p)ppGpp synthesis
The first is the RelA-dependent synthesis. During amino acid starvation, uncharged tRNA molecules bind to the acceptor site on ribosomes, stalling protein synthesis. During this paused protein synthesis, RelA (which is found associated with ribosomes) synthesizes (p)ppGpp from GTP or GDP respectively, in a process that utilizes ATP. The abundance of RelA is relatively low (1/200 ribosomes). However, very large concentrations of (p)ppGpp are produced rapidly because the synthesis of (p)ppGpp results in the dissociation of RelA from the ribosome, allowing the RelA protein to shuttle to another stalled ribosome and repeat the process.
SpoT-dependent (p)ppGpp synthesis
SpoT has distinct active sites that can synthesize or hydrolyze (p)ppGpp
Starvation/shortage of energy triggers conformational change in acyl carrier protein (ACP), which in turn binds to SpoT and shifts balance of activity towards synthesis
The second mechanism for (p)ppGpp synthesis revolves around the function of the SpoT protein. SpoT harbours distinct active sites that can either catalyse the synthesis of hydrolysis of (p)ppGpp. Acyl carrier protein (ACP) is an essential co-factor in fatty acid metabolism, and it physically interacts with SpoT. In instances of fatty acid starvation, ACP undergoes a conformational change that shifts the balance of SpoT activity towards (p)ppGpp synthesis rather than hydrolysis. Glucose starvation, iron starvation and phosphate starvation are also believed to function through the same pathway – modulating SpoT activity.
pppGpp and the open complex during transcription
ALARMONES (p)ppGpp (in conjunction with a co-factor, DksA) destabilizes all open complexes during transcription initiation (especially GC sites)
Because of the higher stability of AT-rich open complexes, they can withstand the destabilising effect of (p)ppGpp, and remain open
> transcriptionally active
Genes repressed by the stringent response have GC-rich ‘discriminator’ sequences within the promoter region
Genes activated by the stringent response, generally have AT-rich ‘discriminator’ sequences
what are the indirect effects of (p)ppGpp on transcription?
Some studies suggest (p)ppGpp reduces the affinity of σ70 for Rpol, increasing the pool of free RNApol that can then complex with alternative sig factors
Suppressing rRNA transcription will free a significant pool of Rpol
(p)ppGpp can modulate the synthesis and/or stability of alternative sigma factors
