Friday, September 14, 2012

ppGpp regulates GTP synthesis by inhibiting Gmk and HprT

Another role for ppGpp was discovered by Allison Kriel and colleagues: it directly regulates GTP levels by interfering with GTP biosynthetic pathway. It is worth saying that inhibitory effects of (p)ppGpp on IMP dehydrogenase and anenylosuccinate synthetase, first enzymes of the guanylate and adenylate pathways, respectively, were discovered by Gallant and colleagues as early as in 1971, so the connection between (p)ppGpp and metabolism of G nucleotides was known long ago.

The cool thing about the paper is the approach they use. There can be loads of potential targets of ppGpp, and loads of proteins will be inhibited by it in vitro. Which ones are the relevant ones? Kriel and colleagues usa a top-down approach: first they get a birds-eye view of starvation by running metabolic and transcriptomic analysis of starved wt and ppGpp0 (i.e. devoid of ppGpp) strains, then identify the ppGpp targets by clustering and pathway analyses, and then follow their predictions up in vitro and an vivo. This is a really powerful approach.

The net result is that (p)ppGpp inhibits several enzymes in the GTP biosynthesis pathway, and by doing quantitative experiments, Kriel et al. identify the primary targets, Gmk and HprT (Fig. 1). All the experiments are done in B. subtilis, and in this bug pppGpp is the major magic spot nucleotide. It is made of GTP, so inhibition of GTP synthesis results in a negative feedback control loop. This loop turns out to be a key component for the control of the GTP levels in the cell.

In  B. subtilis, unlike E. coli, ppGpp does not regulate RNA polymerase directly: the regulation goes via effects on the GTP level. And indeed, a genetic screen performed by Kriel et al. showed that regulation of GTP metabolises by ppGpp, not of polymerase is crucial for the bacterial survival under stress. The have found 37 suppressor mutations leading to survival of the ppGpp0 strain - and most of these turned out to be in the GTP biosynthesis pathway. In the E. coli case, the supressor mutations are usually in the RNA polymerase.

 The negative control of GTP synthesis by ppGpp turned out to be crucial for bacterial well-being - in the ppGpp0 cells high levels of GTP caused cell death, though the mechanism is still unclear (Fig. 1). Kriel et al. proposed several possible explanations: inhibition with ATP-consuming enzymes, excessive up-regulation of the rRNA transcription, effects on dGTP synthesis etc.

Fig. 1: ppGpp's role in B. subtilis survival via regulation of GTP biosynthesis. Figure from Kriel et al.


Kriel et al, Cell (2012) in press

Gallant et al. JBC (1971), 246 (18): 5812-5816, PIMD: 4938039

Krasny and Grouse, EMBO J. (2004), 23(22): 4473-83, PIMD: 15496987

Thursday, September 13, 2012

One more GTPase that binds ppGpp: RF3

The stringent response alarmone ppGpp is very, very similar to GDP: add two more phosphates and you have it... So it is only natural that GTPases mistake the two.

Now Kihira and collegues has added one more to the list - bacterial termination factor 3, RF3. This factor is involved in release of the class one release factors, RF1 and RF2. It binds to the ribosome in the GDP-bound state, exchanges GDP to GTP and kicks the class 1 factors offppGpp:RF3 complex is not as active as GDP:RF3 one, a situation similar to that with initiation factor 2, IF2

The question is is it a specific regulatory mechanism or are GTPases just promiscuous enough? After all, even archael elongation factor 1A, aEF1A, is inhibited by ppGpp as well. And these two never meet in nature...

Kanjee and collegues have recently compiled a list of proteins that ppGpp binds to... or is expected to bind to. GTPases are there, as a whole class of enzymes.


Kihira et al. FEBS J. (2012) in press

Kanjee et al. Mol. Microbiology (2012): 85 (6) 1029-1043 PIMD: 22812515