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Pseudouridine at position 55 in tRNA controls the contents of other modified nucleotides for low-temperature adaptation in the extreme-thermophilic eubacterium Thermus thermophilus.

ABSTRACT: Pseudouridine at position 55 (?55) in eubacterial tRNA is produced by TruB. To clarify the role of the ?55 modification, we constructed a truB gene disruptant (?truB) strain of Thermus thermophilus which is an extreme-thermophilic eubacterium. Unexpectedly, the ?truB strain exhibited severe growth retardation at 50 °C. We assumed that these phenomena might be caused by lack of RNA chaperone activity of TruB, which was previously hypothetically proposed by others. To confirm this idea, we replaced the truB gene in the genome with mutant genes, which express TruB proteins with very weak or no enzymatic activity. However the growth retardation at 50 °C was not rescued by these mutant proteins. Nucleoside analysis revealed that Gm18, m(5)s(2)U54 and m(1)A58 in tRNA from the ?truB strain were abnormally increased. An in vitro assay using purified tRNA modification enzymes demonstrated that the ?55 modification has a negative effect on Gm18 formation by TrmH. These experimental results show that the ?55 modification is required for low-temperature adaptation to control other modified. (35)S-Met incorporation analysis showed that the protein synthesis activity of the ?truB strain was inferior to that of the wild-type strain and that the cold-shock proteins were absence in the ?truB cells at 50°C.

SUBMITTER: Ishida K

PROVIDER: S-EPMC3064792 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Pseudouridine at position 55 in tRNA controls the contents of other modified nucleotides for low-temperature adaptation in the extreme-thermophilic eubacterium Thermus thermophilus.

Ishida Kazuo K Kunibayashi Takashi T Tomikawa Chie C Ochi Anna A Kanai Tamotsu T Hirata Akira A Iwashita Chikako C Hori Hiroyuki H

Nucleic acids research 20101118 6

Pseudouridine at position 55 (Ψ55) in eubacterial tRNA is produced by TruB. To clarify the role of the Ψ55 modification, we constructed a truB gene disruptant (ΔtruB) strain of Thermus thermophilus which is an extreme-thermophilic eubacterium. Unexpectedly, the ΔtruB strain exhibited severe growth retardation at 50 °C. We assumed that these phenomena might be caused by lack of RNA chaperone activity of TruB, which was previously hypothetically proposed by others. To confirm this idea, we replace ...[more]

PMID: 21097467

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Project description:Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel(+); wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNA(Ser) aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).

Dataset Information

Pseudouridine at position 55 in tRNA controls the contents of other modified nucleotides for low-temperature adaptation in the extreme-thermophilic eubacterium Thermus thermophilus.

Publications

Pseudouridine at position 55 in tRNA controls the contents of other modified nucleotides for low-temperature adaptation in the extreme-thermophilic eubacterium Thermus thermophilus.

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