Stringent Response

Stringent R esponse 1891 common bacteria are the result of mutations in rpsL, the gene encoding the ribosomal protein S12 (a protein in the small subunit of the ribosome), and in rrs, the gene encoding 16S ribosomal RNA (rRNA). These mutations are recessive. Since the fast-growing bacteria used as genetic models, such as Escherichia coli and Bacillus subtilis, have several copies of the genes encoding rRNA, most of the early work on streptomycin resistance involved mutations in rpsL (originally named str), which exists as a single copy. Mutants in the gene encoding 16S rRNA conferring streptomycin resistance were not uncovered until genetic techniques were available to manipulate these genes in vitro. Ribosomal protein S12 interacts with a highly conserved structure formed by the 16S rRNA, where streptomycin binds. Apparently certain amino acid changes in S12 lead to an alteration or destabilization of this structure. This in turn affects the binding of streptomycin to the ribosome. Some of these mutations lead to streptomycin resistance, but some lead to streptomycin dependence. As mentioned above, streptomycin itself can increase errors in protein synthesis. Interestingly, some streptomycin-resistant mutants restrict the normal level of certain errors, i.e., the ribosomes in these mutants are hyperaccurate. They also have slowed down translation elongation rates. (Certain mutants in ribosomal protein S4, encoded by rpsD, are also streptomycin resistant and have hyperaccurate ribosomes.) Like streptomycin, these mutants have proved valuable in investigations of translational accuracy. Several different mutations in rpsL are known in enteric bacteria which can lead to streptomycin resistance, but these tend to be clustered at two different regions of the protein: amino acid residues 41 to 45 and 87 to 93. Mutations in similar locations are known in other bacteria. Mutations in the 16S rRNA encoding gene, rrs, which confer resistance to streptomycin have been localized to the region near base 530 and to that near base 915. These regions are part of a putative `accuracy center' of the ribosome. Mutations in the 915 region not only can lead to streptomycin resistance, but also to changes in translational accuracy. This region seems to be involved with proper selection of tRNA at the ribosomal A site. Although the causative agent of tuberculosis, Mycobacterium tuberculosis, has a reasonably large genome (4.4 million bp) it has only a single copy of each rRNA gene. Therefore, in M. tuberculosis, resistance can arise by a mutation in the sole rrs gene (or the sole rpsL gene). One study found that about 10% of the resistant strains of M. tuberculosis isolated from patients have mutations in rrs, while 50% have mutations in rpsL. However, resistance can also arise by

mechanisms other than modification of the target of streptomycin activity, which include uptake and modification of the antibiotic. Although most antibiotics that act by inhibiting protein synthesis are bacteriostatic, streptomycin is bacteriocidal. It is not completely clear why streptomycin kills bacteria, rather than just stopping growth. See also: Antibiotic Resistance; AntibioticResistance Mutants; Resistance to Antibiotics, Genetics of; Ribosomal RNA (rRNA); Ribosomes; Streptomyces

Stringent Response M Cashel doi: 10.1006/rwgn.2001.1250

The bacterial stringent response refers to the many adjustments of gene expression and cell physiology attributable to the accumulation of the (p)ppGpp nucleotides, which are derivatives of GTP (or GDP) bearing pyrophosphoryl substituents on the ribose 30 hydroxyl. The intracellular level of (p)ppGpp is regulated by mechanisms that sense the availability of different nutrients such as amino acids, carbon sources, nitrogen sources, lipids, and phosphate. The best-understood nutrient limitation condition, mediated by the relA gene, involves amino acid deprivation. The stringent response was first noticed as inhibition of stable RNA accumulation occasioned by amino acid starvation in Escherichia coli. The ability of mutants of a single locus to abolish this wild-type `stringent' RNA control phenotype led to calling the mutant behavior a `relaxed response' and the mutant gene relA. Similar mutant phenotypes are widespread among bacteria distantly related to E. coli. In addition to RNA control, many other processes are affected by the stringent response as judged by differential negative or positive mutant effects on regulatory behavior. Negative effects are seen for activities whose functions are presumably superfluous during starvation conditions, such as the synthesis of ribosomes, ribosomal RNA, and transfer RNA. Among functions that can be induced by (p)ppGpp synthesis are synthesis and transport of specific amino acids, accumulation of glycogen and polyphosphate, and induction of the RpoS sigma factor governing stationary phasespecific gene expression. Many of the regulatory outcomes of the stringent response can be viewed as enhancing survival and adaptation to nutritional stress.

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Structural Gene

Using ATP as a pyrophosphate donor to GTP (or GDP) acceptor substrates, the RelA protein catalyzes (p)ppGpp synthesis on ribosomes. The reaction requires that ribosomes be stalled during translation of mRNA for lack of a bound, codon-specified, charged (aminoacylated) tRNA. Catalysis is activated by uncharged cognate tRNA binding to the otherwise vacant ribosomal acceptor site. Predictions that (p)ppGpp synthesis is activated by increased ratios of uncharged/charged tRNA whenever rates of tRNA aminoacylation fail to keep up with the demands of protein synthesis have been verified with aminoacyl-tRNA synthetase mutants when tRNA levels are artificially varied. A causal role for the (p)ppGpp nucleotides in the stringent response can be demonstrated with engineered gene constructs that allow manipulation of (p)ppGpp abundance atwill in cells that are not nutritionally stressed. Cells with an artificially elevated level of (p)ppGpp mimic many of the major regulatory effects seen during a stringent response provoked by amino acid starvation. Regulation of (p)ppGpp levels in response to deprivation of nutrients other than amino acids occurs in strains deleted for relA. Despite the absence of relA function, these starvation protocols elicit responses that share features of the classical stringent response to amino acid limitation. This second source of (p)ppGpp synthesis, in E. coli, is a gene called spoT that encodes a single bifunctional protein having weak (p)ppGpp synthetic activity as well as a specific (p)ppGpp 30 -pyrophosphoryl hydrolase. Although the SpoT protein sequence shows broad homology with the RelA protein, SpoT is not ribosome associated. The regulation of (p)ppGpp accumulation generally involves inhibition of degradation rather than stimulation of synthesis. The best-studied example (carbon source starvation) leads to (p)ppGpp accumulation through severe inhibition of (p)ppGpp hydrolysis. Deleting both the relA and spoT genes of E. coli abolishes detectable (p)ppGpp. Such (p)ppGppo strains appear nearly normal as long as abundant nutrients are provided. However, (p)ppGppo strains fail to grow on otherwise supportive glucose ‡ salts minimal media unless several amino acids are provided. The corresponding biosynthetic pathways are deduced to be (p)ppGpp-dependent. Survival of (p)ppGppo strains is also impaired by nutrient starvation, revealing a protective effect of (p)ppGpp during the stringent response. Although extragenic suppressors of these (p)ppGppo phenotypes map exclusively to genes specifying subunits of the RNA polymerase, the mechanism by which (p)ppGpp inhibits transcription in vitro remains elusive. The stringent response appears to be confined to Eubacteria where specialized roles for (p)ppGpp

range from those found in E. coli to those contributing to pathogenesis (Legionella pneumophila), acid resistance (Lactococcus lactis), adaptive catabolism (Pseudomonas putida), antibiotic production (Streptomyces coelicolor), and quorum sensing for fruiting body development (Myxococcus xanthus). In contrast to most Eubacteria, the genomes of some intracellular parasitic bacteria lack genes with Rel/Spo homology; examples are Rickettsia prowazekii, Treponema pallidum, and Chlamydia trachomatis.

Further Reading

Cashel M, Gentry DM, Hernandez VJ and Vinella D (1996) The stringent response. In: Neidhardt FC et al. (eds) Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn, pp. 1458 ±1496. Washington, DC: ASM Press.

See also: Gene Expression; GTP (Guanosine Triphosphate)

Structural Gene Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.2044

A structural gene is any gene coding for a product (e.g., enzyme, structural protein, tRNA), i.e., any product other than a regulator. See also: Housekeeping Gene

Subcellular RNA Localization T Hazelrigg Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1133

In many types of cells, in diverse species, RNAs are localized to specific cytoplasmic domains. Subcellular RNA localization contributes to the creation of cellular asymmetry by creating spatially unique domains within cells. In some cases, localization of mRNA is coupled to its translational activation, so that only localized transcripts are translated. Together, subcellular RNA localization and localization-dependent translation serve to restrict protein products to specific cellular domains. In recent years considerable advances have been made in understanding the biological functions served by subcellular RNA localization, and the mechanisms behind this localization.