Escherichia coli stringent factor binds to ribosomes at a site different from that of elongation factor Tu or G

Abstract

The binding of Escherichia coli stringent factor to ribosomes has been studied; the reaction depends on 50S and 30S ribosomal subunits and poly(U) as messenger RNA. The ribosome-stringent factor complex is formed at 5-10 mM magnesium acetate; NH4 ions are inhibitory. Binding of the stringent factor to the 70S-mRNA complex is not stimulated by uncharged tRNA. The ribosomal binding site(s) for the stringent factor does not overlap with the one known for the elongation factor Tu (EF-Tu) or G (EF-G). Ribosomes carrying either EF-Tu or EF-G are active in binding the stringent factor; however, they are inactive in synthesizing guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and guanosine 5'-diphosphate 3'-diphosphate (ppGpp). The latter result is due to the blockage of the ribosomal acceptor site by the aminoacyl-tRNA and/or elongation factors. That stringent and elongation factors do not compete for identical ribosomal region(s) is supported by: (1) the reverse experiments where ribosomes charged with the stringent factor are fully active in EF-Tu or EF-G dependent functions; (2) ribosomes that lack the two ribosomal proteins L7 and L12 known to be essential for EF-Tu and EF-G functions bind the stringent factor and are active in synthesizing pppGpp and ppGpp.

Salmonella entericaSerovar Typhimurium BipA Exhibits Two Distinct Ribosome Binding Modes

“…SpoT, together with RelA, regulates ppGpp accumulation in response to cell stress and nutrient deprivation (7). Both SpoT and RelA are thought to associate with and are functionally supported by the ribosome (19,39,42). Recently, CgtA was shown to promote SpoT-dependent activities on the ribosome (24).…”

The binding of the pyrophosphoryl transferase and the elongation factor Tu and G to ribosomes from Escherichia coli

“…These findings are in good agreement with previous experiments in which it was shown that the two acidic proteins L7/L12 from the large ribosomal subunit are not required for the pyrophosphoryl transfer reaction [ 17,18], whereas they are for EF-Tu and EF-G functions [1,2]. The possibility that transferase and elongation factors do not bind to the same but rather to different ribosomes is unlikely, because ribosomes precharged with EF-Tu or EF-G were inactive in the pyrophosphoryl transferase-dependent formation of pppGpp and ppGpp but active in binding the transferase [16]. The inhibition of the synthesis of guanosine Volume 55, number 1 FEBS LETTERS July 1975 Binding of the [3 HI pyrophosphoryl transferase to the 70S-poly U complex and isolation of the complexed ribosomes were carried out as outlined in the legend to table 3.…”

“…polyphosphates was due to the blocking of the acceptor site of the peptidyltransferase center by the elongation factor and/or aminoacyl-tRNA [ 16].…”

“…N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) came from Serva, Heidelberg. EF-Tu [9], EF-G [9] and the pyrophosphoryl transferase [8,16] from E. coli were prepared as described; 70S ribosomes (1 A26o unit a 25 pmol), 50S (1 A26o unit -~ 39 pmol) and 30S (1 A260 unit a 67 pmol) ribosomal subunits were isolated by zonal centrifugation [8] [11 ] and pyrophosphoryl transferase activity [3] were carried out as reported.…”

Ribosomal proteins of Escherichia coli that stimulate stringent-factor-mediated pyrophosphoryl transfer in vitro.

“…SF does not compete either with the binding of elongation factors or with their function (23). Therefore, it is an obvious assumption that SF can remain bound to the 70S ribosome during protein synthesis.…”