Accumulation and turnover of guanosine tetraphosphate in Escherichia coli

A New Relaxed Mutant of Escherichia coli with an Altered 50S Ribosomal Subunit

“…to a growing culture at the appropriate time. Samples (100 pl each) were pipetted into 2 ml of ice-cold 5% trichloroacetic acid; samples were prepared and the radioactivity was counted as described (11). Guanosine tetraphosphate measurements were made on cultures growing in Tris medium (0.35 mM phosphate) to which 20-50 ;pCi/ml of ['2PJortho-phosphate had been added at least one-half generation before the first sampling.…”

“…Guanosine tetraphosphate measurements were made on cultures growing in Tris medium (0.35 mM phosphate) to which 20-50 ;pCi/ml of ['2PJortho-phosphate had been added at least one-half generation before the first sampling. Samples of 50 pul were pipetted at intervals into ice-cold tubes containing 5 ;pl of 13 M formic acid and were chromatographed as described (11). Tests for stringency or relaxedness in suspected mutants or in recombinants were most reliably done as follows: colonies were picked from agar plates, resuspended in separate tubes containing 0.25 ml of the appropriate selective (minimal) growth medium containing 20,ug/ml of jAeucine and 10 pg/ml'of uracil, and grown overnight with shaking.…”

“…Then 1 drop of a mixture containing 500 pg/ml of 5-methyl tryptophan (or was added to each culture. These were incubated with shaking for 50 min, then precipitated with 3 ml of ice-cold 5% trichloroacetic acid; the precipitates were prepared for counting as described (11). Samples prepared in this way were scanned -for the ratio 3H/14C incorporated during the 50-min starvation period.…”

Relationship of the first step in protein synthesis to ppGpp: formation of A(5')ppp(5')Gpp.

“…The accumulation of ppGpp was shown to be due to the recognition of a codon and binding of an uncharged tRNA to the acceptor site on the ribosomes (10,15). It was also shown that ppCpp undergoes a very rapid turnover (12,13). Recently two reports have indicated the formation of ppGpp in eukaryotes, where its accumulation was associated with early stages of differentiation (16,17).…”

“…The discovery by Cashel and Gallant (9) of the two guanine nucleotides, ppGpp and pppGpp, responsible for the stringent response of prokaryotic cells upon amino-acid starvation or a nutritional shift-down in growth medium, was followed by active research into their mechanism of formation (10,11), turnover (12,13), and mode of action (14). The accumulation of ppGpp was shown to be due to the recognition of a codon and binding of an uncharged tRNA to the acceptor site on the ribosomes (10,15).…”