©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Factor for Inversion Stimulation-dependent Growth Rate Regulation of Serine and Threonine tRNA Species (*)

Valur Emilsson (§) , Lars Nilsson (¶)

From the (1)Department of Molecular Biology, Uppsala University, Biomedical Center, Box 590, S-751 24 Uppsala, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have previously shown that the accumulation of 20 tRNA species in Escherichia coli is individually regulated as a function of cellular growth rate. We have also reported that the growth rate regulation of some but not all tRNA species is dependent on the activity of the factor for inversion stimulation (FIS). In present work, we studied the growth rate regulation of the serine- and threonine-accepting tRNA families. We show that the levels of tRNA, tRNA, tRNA, tRNA, and tRNA are reduced in fis cells as the growth rate increases. The accumulation of these tRNA species is reduced 2-5-fold at the fastest bacterial growth rate. The strongest effect is observed for the two minor tRNA species; tRNA and tRNA. In contrast, we find that the accumulation of tRNA, tRNA, and tRNA is similar in wild type and fis bacteria. The data presented provide further evidence for the suggestion that FIS is a stimulating factor that is involved, directly or indirectly, in the high expression level of some tRNA genes at fast bacterial growth rates.


INTRODUCTION

The tRNA multigene family of Escherichia coli consists of 79 genes that are distributed in 41 transcription units, encoding 46 structurally distinct tRNA species (41 different anticodons)(1) . Some tRNA genes are located in the spacer regions within the seven rRNA operons. Others are located in operons coding only for tRNA or are cotranscribed with protein coding sequences. The large number and disparate structure of tRNA operons make the study of tRNA gene expression complicated. In addition to the growth rate control, two mechanisms that actively regulate the accumulation of stable RNA, tRNA and rRNA, have been described: the ppGpp()-dependent stringent control and the more recently described factor for inversion stimulation (FIS)-dependent activation (for reviews see Refs. 2 and 3).

The involvement of ppGpp in repressing the synthesis of stable RNA in response to amino acid or energy source starvation is well documented (2). It has also been suggested that ppGpp is involved in the growth rate regulation of stable RNA synthesis since the level of ppGpp is inversely correlated with the expression level of stable RNA at different growth rates(4) . Furthermore, bacteria that are deficient in degrading ppGpp grow at slower growth rates and have reduced rRNA transcription rates compared with wild type bacteria(5, 6) . However, deletion of the genes that encode the two ppGpp synthetases results in a bacterium (relA, spoT) that, albeit with a slower growth rate, regulates stable RNA synthesis in a normal growth rate-dependent manner(7) . This suggests either that ppGpp is not involved in the growth rate control or alternatively that some other regulatory mechanisms take over in the absence of ppGpp. One such mode of regulation has been described as ribosomal feedback inhibition (8). This phenomenon was observed in cells that overproduced rRNA from plasmids(8, 9) . The expression of plasmid-borne rRNA genes resulted in reduced expression of the chromosomal rRNA and tRNA genes. The ribosomal feedback inhibition mechanism depends on the formation of functionally initiated ribosomes(10) , but the details of this mechanism are not known.

Some stable RNA operons have been shown to be activated by FIS, in vitro or in vivo(11, 12, 13) . FIS-dependent activation is regulated by de novo synthesis of FIS protein, where the fis gene is both auto-regulated and subject to stringent control(14, 15) . Upon dilution of stationary phase cultures in fresh medium or after a nutritional up-shift, the level of the FIS protein increases rapidly and reaches a peak after approximately one generation(15, 16) . The data suggest that FIS is involved in accelerating bacterial growth rate under those conditions.

In order to understand how the composition of the tRNA pools is regulated in E. coli, we have determined the growth rate-dependent level of individual tRNA species in strains where the fis gene is deleted. We have previously studied the tRNA species belonging to the arginine, leucine, and methionine isoacceptor families(17) . In this report, we have studied the growth rate regulation of the serine and threonine-accepting tRNA species in E. coli strain W1485 and in a isogenic fis bacteria. Five tRNA species show a strong dependence on a functional FIS protein for their accumulation. Here, the levels of tRNA are reduced as much as 5-fold in fis bacteria. Expression of the other three tRNA species are identical in wild type and fis bacteria. These data provide support for the suggestion that the FIS protein is involved in the high expression level of many tRNA genes at fast bacterial growth rates.


MATERIALS AND METHODS

Chemicals and Enzymes

[-P]ATP, polynucleotide kinase, and the Hybond-N membranes were purchased from Amersham International (UK). Formamide, phenol, and the components used for the electrophoresis were of ultrapure grade and purchased from International Biotechnologies Inc.

Bacterial Strains

The bacterial strains used for RNA determinations were E. coli W1485 (18) and W1485-fis767 (fis cells)(17, 19) . Cells were grown in M9 medium(20) , supplemented with vitamin B1 (0.01 mM), FeCl (0.03 mM), CaCl (0.1 mM), MgSO (1 mM), and 0.4% of acetate, succinate, or glucose as the carbon source. In addition, cultures were grown in rich medium containing 20 amino acids, purines, and pyrimidines as described by Neidhardt et al. (21) and LB medium containing 0.4% glucose (20).

RNA Isolation

Cells were grown at 37°C with vigorous shaking in a water bath. The cultures were grown in a steady state phase for at least 10 generations as described by Emilsson and Kurland (22) and were harvested on ice at an A of 0.8. The cells were lysed, and the RNA was extracted as described by Emilsson and Kurland(22) .

Electrophoretic Fractionation and Northern Hybridization

Approximately 25 µg of the crude RNA extract was fractionated on horizontal 1.2% agarose gels in TAE buffer (40 mM Tris acetate, pH 7.5, and 1 mM EDTA) and then transferred to Amersham Hybond-N hybridization filters. The filters were hybridized with specific probes for 16 S rRNA and one tRNA species as described previously(17, 22) . The labeled bands were identified by autoradiography, excised, and measured in a liquid scintillation counter for radioactive quantitation. The films were also analyzed by densitometric scanning counter. The tRNA-specific oligodeoxyribonucleotide probes were complementary to the 3`-half of the molecules, i.e. the variable loop, anticodon stem, and anticodon loop sequences of the tRNAs studied (5` to 3`): Ser1: AACCCTTTCGGGTCGCCGGTTTTC; Ser2: GTAGAGTTGCCCCTACTCCGGT; Ser3: CCCCGGATGCAGCTTTTGACC; Ser5: ATACGTTGCCGTATACACAC; Thr1: CTGGGGACCCCACCCCT; Thr2: CCTACGACCTTCGCATT; Thr3: CTGCCGACCTCACCCTT; Thr4: CTGGTGACCTACTGATT. The 16 S rRNA-specific oligodeoxyribonucleotide probe is an 18-mer complementary to the conserved region between positions 562 and 579(23) .


RESULTS

Inactivating the fis gene does not change the growth rate-dependent expression of the rRNA operons(12, 17) . We have therefore used 16 S rRNA as an internal standard and expressed our data as the ratio of tRNA to 16 S rRNA. The target specificity of the eight oligonucleotides used has been tested, showing that the probes are tRNA-specific.()In this study, we find two modes of tRNA regulation. Some tRNA species such as tRNA have reduced expression in fis cells, whereas others like tRNA are unaffected.

The Serine Acceptor tRNA Family

Four tRNA species belong to the serine-accepting tRNA family in E. coli, tRNA, tRNA, tRNA, and tRNA. According to predictions based on the unmodified anticodon, tRNA translates the minor UC(A/G) codons. Transfer RNA translates the two major serine codons UC(U/C), while the two rare serine-accepting tRNAs, tRNA and tRNA, translate the minor serine codons UCG and AG(U/C), respectively.

Accumulation of tRNA, encoded by a single gene in the serT operon(1) , is moderately decoupled from the accumulation of 16 S rRNA in both wild type and fis bacteria (Fig. 2), while the accumulation of the rare tRNA, encoded for by the serU operon(1) , is severely reduced in the absence of FIS (, Fig. 1, and Fig. 2). Here, tRNA accumulation is coupled to the production of 16 S rRNA in wild type cells at various growth rates, while in fis cells the level of tRNA drops 5-fold under the fastest growth condition.


Figure 2: Northern blot analysis of the four tRNA subspecies belonging to the serine acceptor tRNA family. The ratio of tRNA to 16 S rRNA in wild type bacteria at the slowest growth rate is taken as 1. The bars indicate standard errors calculated from six independent experiments. The intracellular concentrations of the tRNA species at the growth rate of 0.5 doublings/h are marked in parentheses with the value for the wild type strain first. A, tRNA (6.3 µM, 4.8 µM); B, tRNA (1.6 µM, 1.4 µM); C, tRNA (1.9 µM, 1.8 µM); D, tRNA (10 µM, 9.5 µM).




Figure 1: Autoradiographs of Northern blot hybridizations of RNA fractionated in 1.2% agarose gels. The hybridizations were performed with probes specific for 16 S rRNA as well as for tRNA (A) and tRNA (B). The RNA preparations were from strain W1485 (lanes1-5) and strain W1485-fis767 (lanes 6-10). The bacteria were grown in M9 + acetate (lanes1 and 6), M9 + succinate (lanes2 and 7), M9 + glucose (lanes3 and 8), complete medium (lanes4 and 9), and LB medium + glucose (lanes5 and 10).



The accumulation of another rare serine acceptor tRNA, tRNA, is found to be coupled to the production of rRNA in wild type bacteria. In contrast, accumulation of tRNA is decoupled from the production of 16 S rRNA in fis cells although not as drastically as we observe for tRNA. Transfer RNA is cotranscribed with four copies of the tRNA gene in the serV operon(1) , and its regulatory response is similar to the previously described tRNA. Finally, the 16 S rRNA-normalized levels of the abundant tRNA, transcribed by two separate operons at positions 20` and 23`, are constant at different growth rates in both wild type and fis cells (, Fig. 1, and Fig. 2). In conclusion, tRNA and tRNA belong to the group of tRNA species that depend on FIS for their growth rate regulation. The tRNA and tRNA species behave as rRNA; they are growth rate-regulated, but the accumulation of the gene products is unaffected by deletion of the fis gene.

The Threonine Acceptor tRNA Family

The threonine isoacceptor tRNA family in E. coli, consists of four species with three unique anticodons(1) . Predictions based on the unmodified anticodon suggest that tRNA and tRNA, with identical anticodons, translate the major threonine codons, AC(U/C), whereas tRNA and tRNA translate the minor ACG and AC(A/G) codons, respectively. The gene for threonine isoacceptor 1 is located in the ribosomal RNA operon, rrnD (1). As expected, the accumulation of tRNA is coupled to the accumulation of 16 S rRNA in both wild type and fis cells (see Fig. 3). In contrast, regulation of tRNA, expressed from the thrW operon(1) , is strongly affected by the deletion of the fis gene ( and Fig. 3). There is already a 35% reduction in the abundance of this tRNA at the slowest growth rate in fis bacteria, and the level decreases additionally 3-fold at the fastest growth rate.


Figure 3: Northern blot analysis of the four tRNA subspecies belonging to the threonine-accepting family. The ratio of tRNA to 16 S rRNA in wild type bacteria at the slowest growth rate is taken as 1. The bars indicate standard errors calculated from six independent experiments. The intracellular concentrations of the tRNA species at the growth rate of 0.5 doublings/h are marked in parentheses with the value for the wild type strain first. A, tRNA (1.6 µM, 1.5 µM); B, tRNA (2.5 µM, 1.6 µM); C, tRNA (3.8 µM, 3.5 µM); D, tRNA(3.5 µM, 2.9 µM).



Two threonine-accepting tRNA species, tRNA and tRNA, are expressed from the thr(U)tufB operon(1) . These tRNA genes are located in the same operon and are therefore expected to be expressed in a similar way at different growth rates. Indeed, in wild type cells the accumulation of both tRNA species is coupled to the accumulation of 16 S rRNA at various growth rates (Fig. 3). In fis cells growing in rich medium, the expression of these two tRNA genes is reduced 2-fold (), which suggests that FIS is required for their high expression level. These results are consistent with the data of Nilsson et al.(11, 16) . However, the present data contradicts the finding by Lazarus and Travers(25) , showing that the absence of FIS does not affect the expression level of the thr(U)tufB operon. Summarizing the regulation of the threonine-accepting tRNA family, tRNA, tRNA, and tRNA depend on FIS for their growth rate regulation. The tRNA is growth rate-regulated, but the accumulation of the gene products is unaffected by deletion of the fis gene.


DISCUSSION

The impetus for this study is to illustrate further the involvement of FIS in the growth rate-dependent regulation of individual tRNA species in E. coli. In a previous study, we found that the growth rate-dependent expression of arginine, methionine, and leucine-accepting tRNA species can be divided into four groups pertaining to the effect of deleting the fis gene(17) . The accumulation rate of one group of tRNA species decreases in the absence of FIS. A second group of tRNA species have been shown to increase their accumulation rate in the absence of FIS. The accumulation rate of a third group of tRNA species is growth rate-regulated but unaltered in fis cells. Thus, the regulatory response to the absence of FIS for these tRNA species is similar to the response found for rRNA(12) . A fourth group contain tRNA species with a weak or undetectable increase in accumulation rate at faster growth rates, thus seeming to be under no growth rate regulation. The accumulation of these tRNA species is also unaffected by the absence of FIS. In the present study, we show that tRNA species within the serine and threonine acceptor families fall into two of these groups (Fig. 1).

tRNA Species with Reduced Accumulation Rates in fis Cells

The growth rate-dependent accumulation of the tRNA, tRNA, tRNA, tRNA, and tRNA species is reduced in fis cells, suggesting that they are, directly or indirectly, activated by FIS and that the expression cannot be fully compensated for by relaxation of the ribosomal feedback system (see below). In fact, the accumulation of the rare tRNA species tRNA and tRNA is severely affected by the deletion of the fis gene, with a 5-fold lower accumulation rate under the fastest growth condition. We have previously shown that also the rare tRNA species tRNA and tRNA are affected by lack of FIS(17) . Thus, in our studies, all rare tRNA species that are growth rate-regulated depend on FIS for this regulation. It has been suggested that two of these rare tRNA species are required for mechanisms other than translation. The cell division mutation ftsM1 has been mapped to the tRNA gene(26) , and the tRNA gene complements a temperature-sensitive dnaY mutation. Since this mutation affects the polymerization phase of DNA replication, tRNA has been implicated in the replication process(27) . It is shown that fis cells are defective in DNA replication and cell division control(28, 29) . FIS binds to the origin of replication (oriC), and it has been suggested that the lack of FIS bound to oriC in fis cells causes the replication and cell division defects. However, a recent study shows that FIS does not stimulate the replication process in vitro(30) . An alternative explanation is that the fis cell defects are caused by reduced levels of tRNA and tRNA. An altered cell morphology, presumably due to the cell division and replication defects, is only seen at fast growth rates of fis cells, when the levels of tRNA and tRNA are most dramatically reduced.()

The expression levels of the three tRNA genes encoding tRNA, tRNA, and tRNA are also reduced when the fis gene is deleted but to a lesser degree than the levels of tRNA and tRNA. The tRNA and tRNA genes are located in the thrU(tufB) operon. This operon was the first to be shown to be regulated by FIS(11) . Using an operon fusion where the tufB gene was replaced with the gene for galactokinase, it was shown that approximately half of the growth rate-dependent increase in the expression of galactokinase depended on the presence of the FIS-binding sequence upstream of the promoter(16) . More recently, Lazarus and Travers (25) have reported that the wild type thrU(tufB) promoter is not FIS-dependent. They ascribed the previous result to a cryptic down-mutation in the promoter region. This discussion is made more complicated by the finding of Vind et al.(31) , showing that the expression of reporter genes from strong promoters on multicopy plasmids is not linear in relation to mRNA levels. We demonstrate that fast growing fis cells contain half the amount of the threonine-accepting tRNA species that is under the control of the thrU(tufB) promoter compared with wild type cells. Our data are in good agreement with previously reported data (16) but in clear contrast to the data of Lazarus and Travers(25) .

tRNA Species with Unaltered Accumulation Rates in fis Cells

The accumulation rates of the Ser1, Ser5, and Thr1 tRNA species increase at faster growth rates. However, the expression level of these RNA species is not reduced by the deletion of the fis gene. These and other tRNA species whose growth rate-dependent regulation responds only mildly to the absence of FIS must therefore be regulated by other means. There is evidence suggesting that at least one mechanism by which these tRNA species are regulated is identical to the ribosomal feedback system, as discussed below.

Regulation of tRNA Expression

It is clear that the accumulation rate of individual tRNA species respond differently to the absence of FIS. What causes these differences? It is a strong correlation between the accumulation rates of different tRNA species within one operon, both in the presence and absence of FIS(17, 22, 24) . We also observed this pattern in the present study. Here, the Thr3 and Thr4 tRNA species, which genes are located in the thrU(tufB) operon, are coupled to the accumulation of 16 S rRNA in wild type cells while their accumulation is decoupled from 16 S rRNA production in fis cells. Also, Ser3 and Arg2 tRNA species are regulated identically in both strains (see Ref. 17 for Arg2 tRNA). Finally, the expression of the Thr1 tRNA gene, located in rrnD together with rRNA genes, is coupled to the expression of rRNA in wild type and fis bacteria at various growth rates. Our data suggest that tRNA accumulation is regulated predominantly at the transcription initiation level.

In the absence of the upstream activating sequences, transcription from the thrU(tufB) promoter is drastically increased in fis cells compared to wild type bacteria(11, 16) . The thrU(tufB) promoter lacking an upstream activating sequence cannot bind FIS in vitro(11, 32) , indicating that the lowered expression from the upstream activating sequence-less construct in wild type cells is not due to an inhibitory binding of FIS to the promoter region. Such a compensatory mechanism is also noted for the rrnB operon ending at position +1(12) , which argues against the involvement of any downstream elements. We suggest that this compensation is caused by the ribosomal feedback system. Evidence exists that ppGpp is not involved in the ribosomal feedback inhibition. Gaal and Gourse (7) have shown that the growth rate-dependent regulation of the rrnB operon is unaffected in cells incapable of producing ppGpp. This seems not to be caused by an increased FIS-dependent activation of the rrnB operon since a minipromoter construct without any FIS binding sites is still growth rate-regulated in ppGpp-less bacteria (33). Furthermore, deletions of one or several rRNA operons result in increased expression of the remaining rRNA operons. This is not accompanied by a decrease in ppGpp levels, suggesting that the ribosomal feedback inhibition is uncoupled from the ppGpp-dependent regulatory mechanism(34) . However, it should be noted that the findings above have been questioned by Hernandez and Bremer(35) , who have found that the rrnB promoter is not growth rate-regulated in cells lacking ppGpp. It is also noticeable that both the ppGpp-dependent repression of stable RNA synthesis and the ribosomal feedback mechanism have been shown to depend on the promoter region only(36, 37, 38) .

We suggest that the combination of FIS-dependent activation and inhibition due to the ribosomal feedback system determines the growth rate-dependent regulation of the tRNA genes. We suggest that the four groups of tRNA species that we find correspond to tRNA species located in operons regulated by 1) FIS, 2) the ribosomal feedback system, 3) both FIS and the ribosomal feedback system, and 4) none of these mechanisms. Genes regulated by FIS alone should have a growth rate-dependent regulation that disappears or is severely reduced in fis cells. Transfer RNA and tRNA belong to this group. Genes regulated by the ribosomal feedback system only should have an increased expression in fis cells. We did not find tRNA species in the threonine and serine families with this regulatory pattern, but we have previously shown that tRNA, tRNA, and tRNA belong to this group(17) . If the gene is regulated by both mechanisms, a relaxation of the ribosomal feedback system should compensate for the absence of FIS, as is the case for the expression from rrnB(12) . In this investigation, tRNA, tRNA, and tRNA illustrate this group. The last group of tRNA species have poor or no growth rate regulation, suggesting that they are neither regulated by FIS nor by the ribosomal feedback system. None of the threonine or serine tRNA species belong to this group, but previous results show that tRNA, tRNA, tRNA, and tRNA have a poor growth rate regulation and the that expression of these tRNA species is unaffected by the absence of FIS(17) . Intermediates between the groups are also found. Several tRNA species such as tRNA, tRNA, tRNA, and tRNA have slightly reduced expression in the absence of FIS ( Fig. 3and Ref. 17), as expected if the ribosomal feedback system only partly compensates for the lack of FIS.

We have demonstrated a strong dependence on a functional FIS protein for the gene expression of several tRNA species under steady state growth rates of bacteria. We find that for tRNA genes that are dependent on FIS for their up-regulation, the requirement for FIS is most significant under rich growth conditions. Furthermore, the collected data of the 20 tRNA species studied so far suggest that the abundance of bulk tRNA is reduced in fis cells at fast growth rates. The limited supply of translating tRNA species and the unbalance in the tRNA pool in fis cells may partly explain the marked differences in the growth rates between wild type and fis bacteria in rich media.

  
Table: Comparison of individual tRNA levels accumulated in wild type and fis bacteria, grown in various medium compositions



FOOTNOTES

*
This work was supported by grants from the Swedish Natural Science Research Council and from the Swedish Cancer Society (to L. N. and C. G. Kurland).The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: University of Buckingham, Clore Laboratory for Life Sciences, Molecular Biology Section, Buckingham MK 18 1EG, United Kingdom.

To whom correspondence should be addressed: Dept. of Molecular Biology, Uppsala University, Biomedical Center, Box 590, S-751 24 Uppsala, Sweden. Tel.: 46 18 17 49 51; Fax: 46 18 55 77 23.

The abbreviations used are: ppGpp, 5`,3`-diphosphate guanosine; FIS, factor if inversion stimulation.

V. Emilsson and L. Nilsson, unpublished data.

V. Emilsson and L. Nilsson, unpublished observation.


ACKNOWLEDGEMENTS

The gift of the MC1000-fis767 clone by R. Johnson and the construction of W1485-fis767 strain by D. Hughes are gratefully acknowledged.


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