Role of a hisU gene in the control of stable RNA synthesis in Salmonella typhimurium

We have previously reported a fivefold reduction in expression of the ilvGEDA operon in a hisU mutant (hisU1820) originally isolated as a histidine regulatory mutant that exhibited derepressed (deattenuated) expression of the his operon. More recently, we have reported that a unitary explanation of the effect of this mutant on amino acid control is complicated by the observation of relaxed control of stable RNA synthesis during carbon/energy source downshifts. In the present study, we report the results of an analysis of the relaxation in control of RNA synthesis in relation to the accumulation of the guanosine polyphosphates, ppGpp and pppGpp. Unexpectedly, we observed that, despite the inability to restrict RNA accumulation upon carbon/energy downshifts, this mutant formed ppGpp at the normal rate. Further, the evidence clearly indicates that the defective control of RNA in this hisU mutant is not owing to an alteration in the spoT gene and that the relA-mediated RNA control is unaltered. However, relaxed RNA synthesis in hisU is suppressed by hyper-elevated levels of ppGpp; thus, an inverse correlation between RNA accumulation and ppGpp level during carbon/energy downshifts is still demonstrable in the hisU mutant. These data led us to the observation that the increased accumulation of stable RNA upon a carbon/energy downshift is apparently the consequence of a hisU-conferred increase in RNA stability.     

Mutant tRNA<Superscript>His</Superscript> Ineffective in Repression and Lacking Two Pseudouridine Modifications

TRANSFER RNA has been implicated in the regulation of a number of amino-acid biosynthetic operons^1–4. Histidyl-tRNA^His has been shown to be involved in regulation of the histidine operon by analysis of six genes (hisO, hisR, hisS, hisT, hisU, hisW), mutation of which causes derepression of the enzymes of the histidine biosynthetic pathway in Salmonella typhimurium^5–7. A class of derepressed mutants (hisR) has only about 55% as much tRNA^His as the wild type⁴ and in the one example sequenced, contains tRNA^HIS with a structure identical to that of the wild type⁸. Studies of mutants of the gene for histidyl-tRNA synthetase (hisS) indicated that the derepressed phenotype was associated with defects in the charging of tRNA^HISin vitro². The amounts of charged and uncharged tRNA^His present in vivo during physiological derepression of the wild type and in the six classes of regulatory mutants, have been determined⁹. This work has shown that repression of the histidine operon is correlated directly with the concentration of charged histidyl-tRNA^Hisin vivo and not with the ratio of charged to uncharged or the absolute amount of uncharged tRNA^His. The derepression observed in mutants, of hisS (the gene for histidyl-tRNA synthetase), hisR (the presumed structural gene for the single species of tRNA^His) and hisU and hisW (genes presumably involved in tRNA modification) may be explained by the lower cellular concentration of charged tRNA^His which these mutants contain.     

Regulation of isoleucine and valine biosynthesis in Salmonella typhimurium: the effect of hisU on repression control.

The control of isoleucine and valine biosynthesis was examined in a hisU mutant of Salmonella typhimurium. It was found that the levels of expression of the ilvEDA operon and the ilvC gene were significantly reduced relative to an isogenic normal strain when grown in unsupplemented medium. In contrast, this hisU mutant exhibited only a slight reduction in total acetohydroxy acid synthase activity relative to that of the wild type. The hisU and hisU⁺ strains were examined to determine their derepressibility upon either leucine, valine or isoleucine limitation. Only during leucine limitation did the hisU strain exhibit impaired derepressibility relative to the hisU⁺ strain. In addition, repression control of threonine deaminase (the ilvA product of the ilvEDA operon) in this hisU mutant was refractory to exogenous supplementation with either leucine or valine. This response is in distinct contrast to that of the normal strain, in which the single addition of leucine or valine results in a significant reduction in the level of threonine deaminase.     

Mutations in domain V of the 23S ribosomal RNA of Bacillus subtilis that inactivate its protein folding property in vitro

The active site of a protein folding reaction is in domain V of the 23S rRNA in the bacterial ribosome and its homologs in other organisms. This domain has long been known as the peptidyl transferase center. Domain V of Bacillus subtilis is split into two segments, the more conserved large peptidyl transferase loop (RNA1) and the rest (RNA2). These two segments together act as a protein folding modulator as well as the complete domain V RNA. A number of site-directed mutations were introduced in RNA1 and RNA2 of B.subtilis, taking clues from reports of these sites being involved in various steps of protein synthesis. For example, sites like G2505, U2506, U2584 and U2585 in Escherichia coli RNA1 region are protected by deacylated tRNA at high Mg²⁺ concentration and A2602 is protected by amino acyl tRNA when the P site remains occupied already. Mutations A2058G and A2059G in the RNA1 region render the ribosome Ery^r in E.coli and Lnc^r in tobacco chloroplast. Sites in P loop G2252 and G2253 in E.coli are protected against modification by the CCA end of the P site bound tRNA. Mutations were introduced in corresponding nucleotides in B.subtilis RNA1 and RNA2 of domain V. The mutants were tested for refolding using unfolded protein binding assays with unfolded carbonic anhydrase. In the protein folding assay, the mutants showed partial to complete loss of this activity. In the filter binding assay for the RNA–refolding protein complex, the mutants showed an extent of protein binding that agreed well with their protein folding activity.     

Mischarging single and double mutants of Escherichia coli sup3 tyrosine transfer RNA

Mischarging mutants of Escherichia coli sup3 tyrosine transfer RNA have been isolated by selecting for suppression of bacterial amber mutations not suppressed by sup3. Five of the mutants have single base changes in the amino acid acceptor stem (A1, A2, U80, U81 and G82). Mutants A1 and A2 are weak thermosensitive suppressors from which thermostable derivatives have been isolated. Some of these derivatives affect the amount of tRNA synthesized but not the sequence (precursor or promoter mutations), and others are double mutants A1U81 and A2U80. The latter mutant does not mischarge. The efficiency of suppression of A1 and A2 can also be increased by recombination events that lead to duplication and triplication of the suppressor gene.The amino acid inserted by some of these mutants at the amber site has been determined. Mutant A1 inserts glutamine, while U81 and A1U81 insert both glutamine and tyrosine.Taken together the results show that the terminal part of the amino acid acceptor stem has an important role in the specificity of aminoacylation by the glutamine and tyrosine synthetase.     

Initiation of Protein Synthesis in Mammalian Cells with Codons Other Than AUG and Amino Acids Other Than Methionine

Protein synthesis is initiated universally with the amino acid methionine. In Escherichia coli, studies with anticodon sequence mutants of the initiator methionine tRNA have shown that protein synthesis can be initiated with several other amino acids. In eukaryotic systems, however, a yeast initiator tRNA aminoacylated with isoleucine was found to be inactive in initiation in mammalian cell extracts. This finding raised the question of whether methionine is the only amino acid capable of initiation of protein synthesis in eukaryotes. In this work, we studied the activities, in initiation, of four different anticodon sequence mutants of human initiator tRNA in mammalian COS1 cells, using reporter genes carrying mutations in the initiation codon that are complementary to the tRNA anticodons. The mutant tRNAs used are aminoacylated with glutamine, methionine, and valine. Our results show that in the presence of the corresponding mutant initiator tRNAs, AGG and GUC can initiate protein synthesis in COS1 cells with methionine and valine, respectively. CAG initiates protein synthesis with glutamine but extremely poorly, whereas UAG could not be used to initiate protein synthesis with glutamine. We discuss the potential applications of the mutant initiator tRNA-dependent initiation of protein synthesis with codons other than AUG for studying the many interesting aspects of protein synthesis initiation in mammalian cells.     

Intragenic suppressors of temperature-sensitive rne mutations lead to the dissociation of RNase E activity on mRNA and tRNA substrates in Escherichia coli

RNase E of Escherichia coli is an essential endoribonuclease that is involved in many aspects of RNA metabolism. Point mutations in the S1 RNA-binding domain of RNase E (rne-1 and rne-3071) lead to temperature-sensitive growth along with defects in 5S rRNA processing, mRNA decay and tRNA maturation. However, it is not clear whether RNase E acts similarly on all kinds of RNA substrates. Here we report the isolation and characterization of three independent intragenic second-site suppressors of the rne-1 and rne-3071 alleles that demonstrate for the first time the dissociation of the in vivo activity of RNase E on mRNA versus tRNA and rRNA substrates. Specifically, tRNA maturation and 9S rRNA processing were restored to wild-type levels in each of the three suppressor mutants (rne-1/172, rne-1/186 and rne-1/187), while mRNA decay and autoregulation of RNase E protein levels remained as defective as in the rne-1 single mutant. Each single amino acid substitution (Gly→Ala at amino acid 172; Phe → Cys at amino acid 186 and Arg → Leu at amino acid 187) mapped within the 5′ sensor region of the RNase E protein. Molecular models of RNase E suggest how suppression may occur.     

Effect of mutations affecting lysyl-tRNAlys on the regulation of lysine biosynthesis in Escherichia coli.

Summary When studying mutants affecting lysyl-tRNA synthetase or tRNA^Lys (hisT, hisW), a lack of correlation is clearly observed between the amount of lysyl-tRNA and the level of derepression of several lysine biosynthetic enzymes. This excludes the possible role of lysyl-tRNA as the specific corepressor of the lysine regulon. However, the level of derepression of DAP-decarboxylase, the last enzyme of the lysine pathway, is very low in the hisT mutant; this indicates that tRNA^Lys is a secondary effector involved in the regulation of the synthesis of this enzyme.Abbreviations DAP diaminopimelate - KRS lysyl-tRNA synthetase - L-lysine tRNA ligase (AMP) (EC6.1.16) - AK III lysinesensitive aspartokinase (EC 2.7.24) - ASA-dehydrogenase aspartic semialdehyde dehydrogenase (EC 1.2.1.10) - DHDP-reductase dihydrodipicolinic acid reductase - DAP-decarboxylase diaminopimelate decarboxylase (EC 4.1.1.20) - AK I threonine-sensitive aspartokinase - HDHI threonine-sensitive homoserine dehydrogenase     

Fur-dependent detoxification of organic acids by rpoS mutants during prolonged incubation under aerobic, phosphate starvation conditions

The activity of amino acid-dependent acid resistance systems allows Escherichia coli to survive during prolonged incubation under phosphate (P_i) starvation conditions. We show in this work that rpoS-null mutants incubated in the absence of any amino acid survived during prolonged incubation under aerobic, P_i starvation conditions. Whereas rpoS⁺ cells incubated with glutamate excreted high levels of acetate, rpoS mutants grew on acetic acid. The characteristic metabolism of rpoS mutants required the activity of Fur (ferric uptake regulator) in order to decrease the synthesis of the small RNA RyhB that might otherwise inhibit the synthesis of iron-rich proteins. We propose that RpoS (σ^S) and the small RNA RyhB contribute to decrease the synthesis of iron-rich proteins required for the activity of the tricarboxylic acid (TCA) cycle, which redirects the metabolic flux toward the production of acetic acid at the onset of stationary phase in rpoS⁺ cells. In contrast, Fur activity, which represses ryhB, and the lack of RpoS activity allow a substantial activity of the TCA cycle to continue in stationary phase in rpoS mutants, which decreases the production of acetic acid and, eventually, allows growth on acetic acid and P_i excreted into the medium. These data may help explain the fact that a high frequency of E. coli rpoS mutants is found in nature.     

Amino acid residues of the Escherichia coli tRNA(m5U54)methyltransferase (TrmA) critical for stability, covalent binding of tRNA and enzymatic activity

The Escherichia coli trmA gene encodes the tRNA(m⁵U54)methyltransferase, which catalyses the formation of m⁵U54 in tRNA. During the synthesis of m⁵U54, a covalent 62-kDa TrmA-tRNA intermediate is formed between the amino acid C324 of the enzyme and the 6-carbon of uracil. We have analysed the formation of this TrmA-tRNA intermediate and m⁵U54 in vivo, using mutants with altered TrmA. We show that the amino acids F188, Q190, G220, D299, R302, C324 and E358, conserved in the C-terminal catalytic domain of several RNA(m⁵U)methyltransferases of the COG2265 family, are important for the formation of the TrmA-tRNA intermediate and/or the enzymatic activity. These amino acids seem to have the same function as the ones present in the catalytic domain of RumA, whose structure is known, and which catalyses the formation of m⁵U in position 1939 of E. coli 23S rRNA. We propose that the unusually high in vivo level of the TrmA-tRNA intermediate in wild-type cells may be due to a suboptimal cellular concentration of SAM, which is required to resolve this intermediate. Our results are consistent with the modular evolution of RNA(m⁵U)methyltransferases, in which the specificity of the enzymatic reaction is achieved by combining the conserved catalytic domain with different RNA-binding domains.     

From one amino acid to another: tRNA-dependent amino acid biosynthesis

Aminoacyl-tRNAs (aa-tRNAs) are the essential substrates for translation. Most aa-tRNAs are formed by direct aminoacylation of tRNA catalyzed by aminoacyl-tRNA synthetases. However, a smaller number of aa-tRNAs (Asn-tRNA, Gln-tRNA, Cys-tRNA and Sec-tRNA) are made by synthesizing the amino acid on the tRNA by first attaching a non-cognate amino acid to the tRNA, which is then converted to the cognate one catalyzed by tRNA-dependent modifying enzymes. Asn-tRNA or Gln-tRNA formation in most prokaryotes requires amidation of Asp-tRNA or Glu-tRNA by amidotransferases that couple an amidase or an asparaginase to liberate ammonia with a tRNA-dependent kinase. Both archaeal and eukaryotic Sec-tRNA biosynthesis and Cys-tRNA synthesis in methanogens require O-phosophoseryl-tRNA formation. For tRNA-dependent Cys biosynthesis, O-phosphoseryl-tRNA synthetase directly attaches the amino acid to the tRNA which is then converted to Cys by Sep-tRNA: Cys-tRNA synthase. In Sec-tRNA synthesis, O-phosphoseryl-tRNA kinase phosphorylates Ser-tRNA to form the intermediate which is then modified to Sec-tRNA by Sep-tRNA:Sec-tRNA synthase. Complex formation between enzymes in the same pathway may protect the fidelity of protein synthesis. How these tRNA-dependent amino acid biosynthetic routes are integrated into overall metabolism may explain why they are still retained in so many organisms.     

Aminoacylation of RNA Minihelices: Implications for tRNA Synthetase Structural Design and Evolution

Abstract

The genetic code is based on the aminoacylation of tRNA with amino acids catalyzed by the aminoacyl-tRNA synthetases. The synthetases are constructed from discrete domains and all synthetases possess a core catalytic domain that catalyzes amino acid activation, binds the acceptor stem of tRNA, and transfers the amino acid to tRNA. Fused to the core domain are additional domains that mediate RNA interactions distal to the acceptor stem. Several synthetases catalyze the aminoacylation of RNA oligonucleotide substrates that recreate only the tRNA acceptor stems. In one case, a relatively small catalytic domain catalyzes the aminoacylation of these substrates independent of the rest of the protein. Thus, the active site domain may represent a primordial synthetase in which polypeptide insertions that mediate RNA acceptor stem interactions are tightly integrated with determinants for aminoacyl adenylate synthesis. The relationship between nucleotide sequences in small RNA oligonucleotides and the specific amino acids that are attached to these oligonucleotides could constitute a second genetic code.     

Identification of the chloroplast adenosine-to-inosine tRNA editing enzyme

Plastids (chloroplasts) of higher plants exhibit two types of conversional RNA editing: cytidine-to-uridine editing in mRNAs and adenosine-to-inosine editing in at least one plastid genome-encoded tRNA, the tRNA-Arg(ACG). The enzymes catalyzing RNA editing reactions in plastids are unknown. Here we report the identification of the A-to-I tRNA editing enzyme from chloroplasts of the model plant Arabidopsis thaliana. The protein (AtTadA) has an unusual structure in that it harbors a large N-terminal domain of >1000 amino acids, which is not required for catalytic activity. The C-terminal region of the protein displays sequence similarity to tadA, the tRNA adenosine deaminase from Escherichia coli. We show that AtTadA is imported into chloroplasts in vivo and demonstrate that the in vitro translated protein triggers A-to-I editing in the anticodon of the plastid tRNA-Arg(ACG). Suppression of AtTadA gene expression in transgenic Arabidopsis plants by RNAi results in reduced A-to-I editing in the chloroplast tRNA-Arg(ACG). The RNAi lines display a mild growth phenotype, presumably due to reduced chloroplast translational efficiency upon limited availability of edited tRNA-Arg(ACG).     

Comparison of DeltarelA strains of Escherichia coli and Salmonella enterica serovar Typhimurium suggests a role for ppGpp in attenuation regulation of branched-chain amino acid biosynthesis

The growth recovery of Escherichia coli K-12 and Salmonella enterica serovar Typhimurium DeltarelA mutants were compared after nutritional downshifts requiring derepression of the branched-chain amino acid pathways. Because wild-type E. coli K-12 and S. enterica serovar Typhimurium LT2 strains are defective in the expression of the genes encoding the branch point acetohydroxy acid synthetase II (ilvGM) and III (ilvIH) isozymes, respectively, DeltarelA derivatives corrected for these mutations were also examined. Results indicate that reduced expression of the known global regulatory factors involved in branched-chain amino acid biosynthesis cannot completely explain the observed growth recovery defects of the DeltarelA strains. In the E. coli K-12 MG1655 DeltarelA background, correction of the preexisting rph-1 allele which causes pyrimidine limitations resulted in complete loss of growth recovery. S. enterica serovar Typhimurium LT2 DeltarelA strains were fully complemented by elevated basal ppGpp levels in an S. enterica serovar Typhimurium LT2 DeltarelA spoT1 mutant or in a strain harboring an RNA polymerase mutation conferring a reduced RNA chain elongation rate. The results are best explained by a dependence on the basal levels of ppGpp, which are determined by relA-dependent changes in tRNA synthesis resulting from amino acid starvations. Expression of the branched-chain amino acid operons is suggested to require changes in the RNA chain elongation rate of the RNA polymerase, which can be achieved either by elevation of the basal ppGpp levels or, in the case of the E. coli K-12 MG1655 strain, through pyrimidine limitations which partially compensate for reduced ppGpp levels. Roles for ppGpp in branched-chain amino acid biosynthesis are discussed in terms of effects on the synthesis of known global regulatory proteins and current models for the control of global RNA synthesis by ppGpp.     

Structural conservation of an ancient tRNA sensor in eukaryotic glutaminyl-tRNA synthetase

In all organisms, aminoacyl tRNA synthetases covalently attach amino acids to their cognate tRNAs. Many eukaryotic tRNA synthetases have acquired appended domains, whose origin, structure and function are poorly understood. The N-terminal appended domain (NTD) of glutaminyl-tRNA synthetase (GlnRS) is intriguing since GlnRS is primarily a eukaryotic enzyme, whereas in other kingdoms Gln-tRNA^Gln is primarily synthesized by first forming Glu-tRNA^Gln, followed by conversion to Gln-tRNA^Gln by a tRNA-dependent amidotransferase. We report a functional and structural analysis of the NTD of Saccharomyces cerevisiae GlnRS, Gln4. Yeast mutants lacking the NTD exhibit growth defects, and Gln4 lacking the NTD has reduced complementarity for tRNA^Gln and glutamine. The 187-amino acid Gln4 NTD, crystallized and solved at 2.3 Å resolution, consists of two subdomains, each exhibiting an extraordinary structural resemblance to adjacent tRNA specificity-determining domains in the GatB subunit of the GatCAB amidotransferase, which forms Gln-tRNA^Gln. These subdomains are connected by an apparent hinge comprised of conserved residues. Mutation of these amino acids produces Gln4 variants with reduced affinity for tRNA^Gln, consistent with a hinge-closing mechanism proposed for GatB recognition of tRNA. Our results suggest a possible origin and function of the NTD that would link the phylogenetically diverse mechanisms of Gln-tRNA^Gln synthesis.     

Magic Spot Metabolism in an Escherichia coli Mutant Temperature Sensitive in Elongation Factor Ts

A temperature-sensitive mutant of Escherichia coli HAK88 which has been shown to have a lesion in elongation factor Ts (EFTs) was studied with repsect to its metabolism of guanosine 5′-diphosphate, 2′(3′)-diphosphate (ppGpp) and the associated failure of ribosomal ribonucleic acid (rRNA) accumulation at the nonpermissive temperature. Results reported here show that (i) when EFTs is nonfunctional, a full complement of charged transfer RNA (tRNA) cannot prevent accumulation of ppGpp (magic spot) and the stringent failure of rRNA accumulation; (ii) chloramphenicol prevents magic spot (MS) formation and the stringent response not by increasing the percentage of charged tRNA, but possibly by somehow interfering directly with the synthesis of MS; and (iii) tetracycline can lead to MS disappearance without resumption of RNA synthesis. Thus, the absence of MS and the presence of a functional RNA polymerase and charged tRNA are not sufficient to support rRNA accumulation in vivo. An additional element in the regulatory system is suggested.     

The methylation of transfer ribonucleic acid during regeneration of the liver

Transfer ribonucleic acid¹ is methylated after the molecule is synthesized; at least eight enzymes are involved in the transfer of methyl groups (derived from methionine). The time courses of methylation and synthesis of tRNA during rat liver regeneration have been compared in an in vivo radioisotopic study, using 6-orotic acid-¹⁴C and ³H-methyl-L-methionine as precursors in double label pulses. Liver regeneration is a synchronized system in which biochemical events of the cell cycle are separable. Transfer RNA methylation increase precedes by several hours tRNA synthesis during regeneration, although the curves overlap. A ratio of the relative rate of methylation to the relative rate of synthesis has been made; that curve positively correlates with the rise and fall of protein synthesis during regeneration. It is clear that methylation and synthesis of tRNA are only weakly coupled; changing methyl content of the tRNA "pool" resulting from differential tRNA methylase and polymerase activities may regulate the rate of protein synthesis in the cell cycle at the translational level. The "pool sizes" of uridine monophosphate (UMP) and S-adenosylmethionine (SAM) were measured indirectly; UMP and SAM were isolated from perchloric acid supernatants and their specific activities were computed. Differential changes in radioactivity available to tRNA methylases and polymerases are not a source of artifact. That is, the control of both the synthesis and methylation of tRNA is at the enzyme level in vivo, rather than at some enzymatic step prior to those enzymatic reactions.