Hypomodification of the Wobble Base in tRNA^Glu, tRNA^Lys, and tRNA^Gln Suppresses the Temperature-Sensitive Phenotype Caused by Mutant Release Factor 1

Media, strains, and plasmids.The strains used in this study are listed in Table 1. Media and genetic procedures were those described by Miller (23). Where appropriate, the media contained 25 μg/ml tetracycline (Tc), 200 μg/ml ampicillin, 50 μg/ml kanamycin, or 20 μg/ml chloramphenicol (Cm). Unless otherwise stated, cysteine and methionine were added to final concentrations of 100 μM and 200 μM, respectively (24).

Construction of strains.A mini-Tn10 pool in MG1655 was isolated using the system described by Kleckner et al. (17).

To construct strains IIG1 and IIG2, a P1 lysate grown on strain MRA532 was used to transduce AD16, with selection for Tc^r. Transductants were scored for slow/fast growth and ability to grow on M9 minimal plates. One fast-growing isolate, which grew on M9 minimal medium without cysteine, was named IIG1, and one slow-growing clone, which could not grow without cysteine, was named IIG2.

Construction of plasmid pIIG1.Procedures for plasmid construction and transformation were those described by Sambrook et al. (30). A piece of the cysB gene covering the insertion site for IS10 was amplified from MG1655 using primers CysB1 (5′-TAAATCGTATTAGTCACCCGCCAGG-3′) and GI42ned (5′-CAGCCCTAACCGGACGTAAGTTTTA-3′). The amplified fragment was cloned into the pDrive vector (Qiagen), and the sequence was confirmed.

PCR and sequence analysis.Inverted PCR was performed as described by Higashitani et al. (12).

PCR was done using Ready-To-Go PCR beads (GE Healthcare). Sequence analysis was done by Eurofins MWG Operon (Germany).

β-Galactosidase assay.Bacteria were grown at 37°C in M9 minimal medium supplemented with all 20 amino acids (24). The assay was performed as described in reference 34.

Map position of the suppressor mutation.To increase knowledge about the function of RF1 and the termination process, we have studied a mutant allele of RF1 that causes a temperature-sensitive growth phenotype (29). This mutant was used to isolate a collection of independent Ts⁺ suppressor mutants at 42°C (14). One of these suppressor mutants, strain MRA475, was found not to grow on M9 minimal plates. This feature was used to isolate a mini-Tn10 linked to the suppressor locus. A P1 lysate grown on a mini-Tn10 pool (see Materials and Methods) was transduced to strain MRA475, with selection for growth on M9 minimal plates supplemented with Tc. The few transductants obtained were found to be Ts, presumably as a consequence of loss of the suppressor mutation. One clone, MRA533, was kept for further analysis. The frequency of cotransduction with the suppressor was found to be about 0.8, which corresponds to a distance of approximately 0.15 min (33). To determine the position of Tn10, inverted PCR was done from the ends of the transposon, and the resulting products were sequenced. It was found that Tn10 was inserted into the yciS open reading frame. About 0.15 min away from yciS is the cysB gene, and since strain MRA475 cannot grow on minimal medium, cysB was a potential locus for the suppressor mutation. Addition of cysteine to M9 minimal plates enabled strain MRA475 to grow. The cysB gene codes for a transcription factor for the cys regulon (18). To determine the precise position of the suppressor mutation, the region covering topA-cysB was sequenced, and an IS10 element was found in the cysB sequence, with nucleotides 261 to 269 duplicated as an insertion site. The insertion probably leads to loss of expression of the CysB protein.

Genetic evidence that lack of CysB causes the suppressor phenotype.To address the question whether the observed suppressor phenotype is due to a loss of CysB activity, strain MRA475 was transformed with a plasmid-borne wild-type copy of the cysB gene, pCa24N, strain JW1267 (16). The transformants were tested for growth at 37°C and 42°C. As expected, they grew fast at 37°C and had regained the Ts phenotype, which indicates that loss of the CysB protein is responsible for the suppressor phenotype (data not shown).

To verify that the insertion of IS10 causes the suppressor phenotype, a marker rescue experiment was performed. A fragment of the wild-type cysB gene (788 bp) was amplified by using primers flanking the region where the IS10 is inserted. The fragment was cloned, creating plasmid pIIG1, which was transformed into strain MRA475. The transformed strain was plated onto M9 minimal plates with ampicillin to select for a Cys⁺ phenotype. Transformants were tested for growth at 37°C and 42°C and were found to be Ts. If the insertion of IS10 causes the suppressor phenotype, the Ts phenotype can be restored only by homologous recombination between the chromosome and the fragment on the plasmid. A Ts clone was sequenced, and indeed, we found that recombination had taken place and the IS10 was lost from the chromosome.

Next, we combined the prfA1 mutation with an inactivated cysB gene to confirm the suppressor phenotype. To do this, we used strain JWK1267 from the Keio collection, carrying the cysB gene replaced by a Cm^r cassette (4), and transduced it using a phage lysate grown on strain MRA7 (prfA1), selecting for Tc^r. Strain JWK1267 (ΔcysB) grows slowly, but faster than strain MRA475 (prfA1 cysB::IS10), on Luria broth agar (LA) plates. The transductants were tested for growth at 42°C, and two classes were found in accordance with the coupling frequency between prfA1 and Tn10. One class grew like the recipient both at 37°C and at 42°C, whereas the other class grew like MRA475 at both temperatures, i.e., more slowly than JWK1267. We hypothesize that only Tn10 was transduced in the former class, while both Tn10 and prfA1 were transduced in the second class. This result was verified by transduction of Tn10 from each class to MG1655 with selection for Tc^r. As expected, the first class yielded only Ts⁺ transductants, while the other class yielded Ts transductants with a 40% coupling frequency to Tn10. Hence, we conclude that lack of the CysB protein does suppress the Ts phenotype caused by prfA1.

Does CysB affect regulation of the gene for RF1?CysB is a transcription factor for the cys regulon; it functions as an activator of most operons but is an autoregulatory repressor (18). It was shown previously that overexpression of mutant RF1 suppresses the Ts phenotype (28). Therefore, we investigated whether the CysB protein also represses the gene for RF1. The prfA gene is the second gene in an operon, preceded by the hemA gene, and there are 41 nucleotides between the two genes, which allows for regulatory elements. Therefore, to test our hypothesis, we used strain AD16, which carries the beginning of the hemA operon, starting from the promoters in front of the hemA gene up to the beginning of the prfA1 gene, fused to lacZ. Such a construct measures the expression of RF1 both transcriptionally and translationally (9). cysB::IS10 was transduced to strain AD16, creating strains IIG1 and IIG2.

β-Galactosidase activity was measured in strains IIG1 and IIG2. The results showed that expression in the two strains was approximately the same (5.8 × 10⁻³ and 4,8 × 10⁻³ U, respectively). We conclude that CysB is not involved in the regulation of prfA gene expression, and hence, suppression is not a consequence of overexpression of the mutant RF1 protein.

The suppressor phenotype in MRA475 disappears when excess cysteine is added.We wanted to test whether the suppressor phenotype is a consequence of a lack of cysteine. It should be noted that all our experiments were done using LA plates, which contain an undetermined amount of cysteine. Therefore, we added cysteine to a final concentration of 100 μM (in addition to what was already in the plate) to LA plates and tested the growth of strains MG1655, MRA7, and MRA475 at 42°C. That concentration is based on the optimal concentration determined by Neidhardt et al. (24). It was found that the suppressor phenotype in strain MRA475 was not affected (Fig. 3A and 4), while growth at 37°C was faster (data not shown). All growth experiments described in this work were done on plates, because many of the mutants grow so slowly that the risk of faster-growing revertants obscuring the experimental results is very high in liquid cultures. In a further experiment, we added increasing amounts of cysteine to the plates. When 200 μM cysteine was added, the suppressor phenotype was weakened, and with the addition of 300 μM cysteine, the suppressor phenotype disappeared (Fig. 3B and C, respectively). Growth at 37°C was not changed from that with the addition of 100 μM cysteine. This result led us to speculate that it is not the lack of cysteine per se that causes the suppressor phenotype, but something dependent on cysteine. Cysteine is an important donor of sulfur to several enzymes; it also donates the sulfur needed for the formation of methionine and thus for that of S-adenosyl-methionine (18, 31). To test whether insufficient amounts of methionine or S-adenosyl-methionine cause the suppressor phenotype, we added increasing amounts of methionine with or without 100 μM cysteine. The suppressor phenotype was not affected by increasing amounts of methionine (data not shown). Hence, we suggest that it is the lost ability to donate sulfur to some key component(s), other than methionine, that causes the suppression. One such pathway is the thiolation of tRNA (Fig. 2).

• Open in new tab
• Download powerpoint

FIG. 3.

Test of the Ts phenotype at different cysteine concentrations. Strains MG1655 (wild type) (sector 1), MRA475 (prfA1 cysB::IS10) (sector 2), and MRA7 (prfA1) (sector 3) grown on LA plates at 42°C supplemented with either 100 μM (A), 200 μM (B), or 300 μM (C) cysteine. The plates were incubated for about 36 h.

• Open in new tab
• Download powerpoint

FIG. 4.

Test of the Ts phenotype on LA plates at 42°C in strains with the prfA1 mutation in combination with different mutations affecting the synthesis of thiolated nucleosides. Sectors: 1, IIG46 (ΔmiaB prfA1); 2, IIG44 (ΔthiI prfA1); 3, MG1655 (wild type); 4, MRA8 (prfA1); 5, MRA514 (mnmE prfA1); 6, IIG16 (ΔcysB); 7, IIG28 (ttcA1 prfA1); 8, IIG29 (mnmA2 prfA1). The plates were incubated for about 36 h.

Which thiolation(s) of tRNA causes the suppressor phenotype?The synthesis of all thiolated nucleosides in tRNA depends on the cysteine desulfurase enzyme (IscS), encoded by the iscS gene (19). Sulfur from cysteine is transferred to IscS, creating a persulfide, and the sulfur is, in turn, involved in the synthesis of thiolated nucleosides (Fig. 2). To investigate whether it is the lack of one or more tRNA thiolations that causes the suppressor phenotype, we tested whether an IscS mutant could suppress the Ts suppressor phenotype. We tried to combine a mutation in iscS with the prfA1 mutation but failed. A strain with a mutation in iscS grows very poorly, which makes it essentially impossible to manipulate genetically.

The IscS protein donates sulfur to two distinct pathways for the thiolation of tRNA: one is a [Fe-S] protein-independent pathway and the other a [Fe-S] protein-dependent pathway (Fig. 2). To test whether the thiolation of tRNA is involved in the suppressor phenotype, strains with mutations in genes affecting each modification—miaB (s²C), thiI (s⁴U), ttcA (ms²A), and mnmA (s²U)—were tested for their suppressor ability. A phage lysate grown on strain MRA7 (prfA1) was used to transduce strains GRB1776 (ttcA1), JW0413 (ΔthiI), and JW0658 (ΔmiaB), with selection for Tc^r. When the transductants were tested for growth at 42°C, we found that 40% of the transductants with ttcA1, ΔthiI, or ΔmiaB strains as the recipient were Ts, reflecting the cotransduction frequency between prfA1 and Tn10. These transductants had presumably received the prfA1 allele, which is not suppressed. To analyze the s² group of mnm⁵s²U, another phage lysate, grown on strain TH178 (mnmA2), was used to transduce strain MRA8 (prfA1), again with selection for Tc^r. The distance between mnmA2 and the transposon is 1.1 min, and 0.15% of the transductants were Ts⁺, which correlates well with the distance between the two markers. This indicates that the lack of the s²U modification causes the suppressor phenotype (Fig. 4). The presence of the mnmA2 mutation was confirmed by transductions (data not shown).

The mnm⁵s²U modification.Transfer of sulfur from IscS to MnmA for the synthesis of mnm⁵s²U is a multistep process involving five enzymes: TusA, TusB, TusC, TusD, and TusE (Fig. 2). To confirm the result discussed above, a mutation in any of these five genes should lead to suppression of the Ts phenotype. For this reason, we transduced strain JW3305 (ΔtusB) with a P1 lysate grown on strain MRA7 (prfA1), with selection for Tc^r. The Ts phenotype was tested, and as expected, the mutation in tusB could suppress the Ts phenotype caused by the mutated allele of RF1. That the prfA1 allele is present was shown by transducing it out to a wild-type strain (data not shown).

Since our data point to the lack of the s² modification of the wobble U base as the suppressor, we tested whether lack of the mnm⁵ modification, with s² maintained, would suppress the Ts phenotype. This experiment can be done because the two groups are added independently of each other (6). The MnmE enzyme is necessary for the synthesis of the mnm⁵ group (10); therefore, strain MRA514 (prfA1 mnmE) was tested at 42°C and found to be Ts⁺ (Fig. 4). Hence, the lack of the mnm⁵ group also leads to suppression of the Ts phenotype.