Genetic analysis of the invariant residue G791 in Escherichia coli 16S rRNA implicates RelA in ribosome function.
Previous studies identified G791 in Escherichia coli 16S rRNA as an invariant residue for ribosome function. In order to establish the functional role of this residue in protein synthesis, we searched for multicopy suppressors of the mutant ribosomes that bear a G-to-U substitution at position 791. We identified relA, a gene whose product has been known to interact with ribosomes and trigger a stringent response. Overexpression of RelA resulted in the synthesis of approximately 1.5 times more chloramphenicol acetyltransferase (CAT) protein than could be synthesized by the mutant ribosomes in the absence of RelA overexpression. The ratio of mutant rRNA to the total ribosome pool was not changed, and the steady-state level of CAT mRNA was decreased by RelA overexpression. These data confirmed that the phenotype of RelA as a multicopy suppressor of the mutant ribosome did not result from the enhanced synthesis of mutant rRNA or CAT mRNA from the plasmid. To test whether the phenotype of RelA was related to the stringent response induced by the increased cellular level of (p)ppGpp, we screened for mutant RelA proteins whose overexpression enhances CAT protein synthesis by the mutant ribosomes as effectively as wild-type RelA overexpression and then screened for those whose overexpression does not produce sufficiently high levels of (p)ppGpp to trigger the stringent response under the condition of amino acid starvation. Overexpression of the isolated mutant RelA proteins resulted in the accumulation of (p)ppGpp in cells, which was amounted to approximately 18.2 to 38.9% of the level of (p)ppGpp found in cells that overexpress the wild-type RelA. These findings suggest that the function of RelA as a multicopy suppressor of the mutant ribosome does not result from its (p)ppGpp synthetic activity. We conclude that RelA has a previously unrecognized role in ribosome function.
Kim
Hong-Man
HM
Department of Life Science, Chung-Ang University, Dongjak-Hu, Seoul, Republic of Korea.
Ryou
Sang-Mi
SM
Song
Woo-Seok
WS
Sim
Se-Hoon
SH
Cha
Chang-Jun
CJ
Han
Seung Hyun
SH
Ha
Nam-Chul
NC
Kim
Jae-Hong
JH
Bae
Jeehyeon
J
Cunningham
Philip R
PR
Lee
Kangseok
K
D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006159:Guanosine Tetraphosphate / Q000378:metabolism; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D012329:RNA, Bacterial / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012270:Ribosomes / Q000201:enzymology* / Q000235:genetics; D013489:Suppression, Genetic
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D006159:Guanosine Tetraphosphate; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases