The Stringent Response

Question 1

Bacterial metabolism is regulated by

Answer 1

small molecules

• secondary messengers: ppGpp + cyclic di-GMP
• quorum sensing molecules (AHLs)

Question 2

secondary messengers are

Answer 2

molecules produced in response to a stimulus and then detected by something else to mediate a response

Question 3

The stringent response causes a

Answer 3

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

Question 4

ppGpp is an (3)

Answer 4

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

Question 5

how do cells sense a shortage of an amino acid

Answer 5

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

Question 6

pppGpp is made by

Answer 6

RelA using pppGpp synthase

pppGpp converted back to ppGpp by pppGpp phosphohydrolase

Question 7

RelA is an enzyme on

Answer 7

approx 1 in 200 ribosomes

RelA is activated when an uncharged tRNA enters the A site of the ribosome

Question 8

SpoT can also

Answer 8

make and get rid of pppGpp

SpoT also responds to C, P and Fe starvation and oxidative stress

Question 9

pppGpp binds to

Answer 9

RNA polymerase

stops transcription of rRNA and tRNA, ribosomal proteins, cell wall production and DNA replication

Question 10

pppGpp turns on

Answer 10

stress proteins
amino acid biosythesis pathways
recycling proteolases
stationary phase sigma factors

Question 11

What is particularly sensitive to pppGpp in rRNA and tRNA?

Answer 11

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

Question 12

How does ppGpp work?

Answer 12

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

Question 13

AT rich sequences can be

Answer 13

activated by pppGpp

as transcription slows, more rRNA in the cell = more AT rich sequences transcribed = more stringent response proteins

Question 14

RNA pol is a major transducer of

Answer 14

pppGpp signal

Question 15

Outside reading info

Answer 15

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

Question 16

RelA-dependent (p)ppGpp synthesis

Answer 16

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.

Question 17

SpoT-dependent (p)ppGpp synthesis

Answer 17

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.

Question 18

pppGpp and the open complex during transcription

Answer 18

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

Question 19

what are the indirect effects of (p)ppGpp on transcription?

Answer 19

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