Synthesis of Stable RNA in Stringent Escherichia coli Cells in the Absence of Charged Transfer RNA

Abstract

It has been possible to demonstrate the complete absence of either charged tRNAGlu or charged tRNAVal at 420 by the use of two stringent strains of E. coli, one temperature-sensitive for glutamyl-tRNA synthetase and the other temperature-sensitive for valyltRNA synthetase. In both strains, stable RNA synthesis ceases, and guanosine tetraphosphate accumulates upon incubation at the nonpermissive temperature. Unique among a series of antibiotics tested, only tetracycline was able to stimulate stable RNA synthesis and to cause disappearance of the guanosine nucleotide. In this regard tetracycline and the "relaxed" gene product appear to be analogous.The synthesis of stable RNA is greatly diminished when Escherichia coli is unable to synthesize protein as the result of either a nutritional or mutational block. This linkage between protein and RNA synthesis has been termed the stringent response, and is directly or indirectly dependent upon a functional rel+ allele. Mutants of the rel+ locus have been isolated (1, 2) and are referred to as "relaxed." Phenotypically, a mutation in the ret locus uncouples the stringent response; i.e., it permits RNA synthesis to continue in the absence of protein synthesis. Similarly, when antibiotics that are known to block protein synthesis at the level of the ribosome are added to a stringent strain, RNA synthesis is uncoupled from protein synthesis. It has been suggested that this latter response is dependent upon the accumulation of charged tRNA whose use in protein synthesis has been spared by the presence of the antibiotic (3). Consequently, it was suggested that for RNA synthesis to occur in a stringent strain, a full complement of charged tRNAs and N-formylmethionyl-tRNA (4, 5) are required.More recently, Cashel and Gallant (6-8) identified two unusual guanosine nucleotides, guanosine tetraphosphate and guanosine pentaphosphate (MSI and MSII, respectively), that accumulate during the inhibition of protein synthesis in a stringent strain, but not in a relaxed strain. It was suggested (8) that accumulated ppGpp and/or ppGppp inhibit(s) stable RNA synthesis. Recently, it was reported that ppGpp inhibits the initiation of rRNA synthesis in vitro (9).The question then remains: is it possible to separate the antiobiotic-induced accumulation of charged tRNA from the synthesis of stable RNA in trivo? If such a distinction can be made, it will be possible to determine the relationship between the relaxed phenotype and the antibiotic-induced accumulation of stable RNA. In this communication, we report that tetracycline is unique among several antibiotics tested in that it can stimulate stable RNA synthesis in a stringent strain of E. coli in the complete absence of either charged valyl-or glutamyltRNA; thus, tetracycline presumably can stimulate RNA synthesis in the absence of any charged tRNA species. Thus, tetracycline seems to have a basic similarity to the rel gene product with respect to its mode of action in the absence of charged tRNA. We also show that in the presence ...

Occurrence of the regulatory nucleotides ppGpp and pppGpp following induction of the stringent response in staphylococci

“…Recent studies show that chloramphenicol binds to the ribosomal A site in E. coli (23). However, the precise mechanism by which chloramphenicol prevents (p)ppGpp synthesis is not clear, and studies of the effects of chloramphenicol on temperature-sensitive valyl tRNA synthetase mutants under different conditions draw conflicting conclusions (8,20). This may therefore indicate possible differences between the effective amino acid starvation produced in such mutants and the inhibition of the isoleucyl enzyme by mupirocin.…”

Effect of variation of charged and uncharged tRNA(Trp) levels on ppGpp synthesis in Escherichia coli

“…Simultaneously, 32P (carrier free; 50 ,uCi/ml; Amersham) was added to each culture. Ampicillin and tetracycline were omitted at the time of resuspension, since tetracycline is known to be an inhibitor of ppGpp synthesis (18). Even though our strain was Tetr, we still observed reduced ppGpp synthesis in the presence of tetracycline (data not shown).…”

Characterization of the stringent and relaxed responses of Streptococcus equisimilis

“…In E. coli, tetracycline inhibits the synthetic activity of RelA by blocking the binding of uncharged tRNA to the ribosome acceptor sites (22). Figure 3 shows that tetracycline caused the accumulated (p)ppGpp to disappear, following first-order kinetics.…”