共查询到20条相似文献,搜索用时 15 毫秒
1.
PrrC-anticodon nuclease: functional organization of a prototypical bacterial restriction RNase 总被引:2,自引:0,他引:2
The tRNALys anticodon nuclease PrrC is associated in latent form with the type Ic DNA restriction endonuclease EcoprrI and activated by a phage T4-encoded inhibitor of EcoprrI. The activation also requires the hydrolysis of GTP and presence of dTTP and is inhibited by ATP. The N-proximal NTPase domain of PrrC has been implicated in relaying the activating signal to a C-proximal anticodon nuclease site by interacting with the requisite nucleotide cofactors [Amitsur et al. (2003) Mol. Microbiol., 50, 129–143]. Means described here to bypass PrrC's self-limiting translation and thermal instability allowed purifying an active mutant form of the protein, demonstrating its oligomeric structure and confirming its anticipated interactions with the nucleotide cofactors of the activation reaction. Mutagenesis and chemical rescue data shown implicate the C-proximal Arg320, Glu324 and, possibly, His356 in anticodon nuclease catalysis. This triad exists in all the known PrrC homologs but only some of them feature residues needed for tRNALys recognition by the Escherichia coli prototype. The differential conservation and consistent genetic linkage of the PrrC proteins with EcoprrI homologs portray them as a family of restriction RNases of diverse substrate specificities that are mobilized when an associated DNA restriction nuclease is compromised. 相似文献
2.
3.
Birthe Meineke Beate Schwer Raffael Schaffrath Stewart Shuman 《Nucleic acids research》2011,39(2):687-700
tRNA damage inflicted by the Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies an antiviral response to phage T4 infection. PrrC homologs are present in many bacterial proteomes, though their biological activities are uncharted. PrrCs consist of two domains: an N-terminal NTPase module related to the ABC family and a distinctive C-terminal ribonuclease module. In this article, we report that the expression of EcoPrrC in budding yeast is fungicidal, signifying that PrrC is toxic in a eukaryon in the absence of other bacterial or viral proteins. Whereas Streptococcus PrrC is also toxic in yeast, Neisseria and Xanthomonas PrrCs are not. Via analysis of the effects of 118 mutations on EcoPrrC toxicity in yeast, we identified 22 essential residues in the NTPase domain and 11 in the nuclease domain. Overexpressing PrrCs with mutations in the NTPase active site ameliorated the toxicity of wild-type EcoPrrC. Our findings support a model in which EcoPrrC toxicity is contingent on head-to-tail dimerization of the NTPase domains to form two composite NTP phosphohydrolase sites. Comparisons of EcoPrrC activity in a variety of yeast genetic backgrounds, and the rescuing effects of tRNA overexpression, implicate tRNALys(UUU) as a target of EcoPrrC toxicity in yeast. 相似文献
4.
5.
Larry C. Waters James R. Burke M.P. Stulberg 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,696(2):201-207
Significant amounts of three tRNAs are associated with the 70 S RNA of avian myeloblastosis virus (AMV). The temperatures at which they are half dissociated from the 70 S RNA in 50 mM NaCl and their respective quantities relative to 35 S RNA are: tRNAArg, 51°C, 1.6; tRNALys, 57°C, 0.7 and tRNATrp, 76°C, 1.0. Possible functions for the non-primer tRNAs (tRNAArg and tRNALys) were evaluated by determining the effect of their thermal dissociation on: (a) conversion of 70 S to 35 S RNA, (b) capacity of 70 S and/or 35 S RNA to be translated in vitro, and (c) capacity of 70 S and/or 35 S RNA to be reverse transcribed in vitro. Conversion of 70 S to 35 S RNA occurred with a tm of 56°C and is consistent with the hypothesis that tRNALys might be involved in joining two 35 S RNA subunits to form the 70 S RNA complex. There was no indication that the association of either tRNAArg or tRNALys influenced the rate or quality of translation of 70 S or 35 S RNA. A decrease in the rate at which 70 S RNA is transcribed occurs in parallel with the dissociation of tRNAArg and tRNALys. 相似文献
6.
Cellular changes have been monitored during the suppression, mediated by the overproduction of tRNALys, of thermosensitivity in Escherichia coli strain AA7852 carrying a mutation in peptidyl-tRNA hydrolase (Pth) encoded by the pth(Ts) gene. The presence in AA7852 cells of a plasmid bearing lysV gene helped to maintain low levels of the unstable Pth(Ts) protein and to preserve the viability of the mutant line at 41°C whereas plasmids bearing other tRNA genes were ineffective. At 32°C the excess of tRNALys did not alter the percentages of the free-, charged- or peptidyl-tRNALys species compared with those found in strains that did not overproduce tRNALys. At 41°C, however, despite increases in the level of peptidyl-tRNALys, the excess tRNALys helped to maintain the concentration of charged-tRNALys at a level comparable with that found in non-overproducer cells grown at a permissive temperature. In addition, the excess tRNALys at 41°C provoked a reduction in the concentrations of various peptidyl-tRNAs, which normally accumulate in pth(Ts) cells, and a proportional increase in the concentrations of the corresponding aminoacyl-tRNAs. The possible mechanism of rescue due to the overexpression of tRNALys and the causes of tRNALys starvation in pth(Ts) strains grown at non-permissive temperatures are considered. 相似文献
7.
8.
Yan Lu Tiehui Xiao Feng Zhang Yanming Chen Yuqi Liu Yang Li Yun dai Chen Zongbin Li Minxin Guan 《Biochemical and biophysical research communications》2014
Mitochondrial dysfunction has been potentially implicated in both human and experimental hypertension. We performed the mutational analysis of tRNALys gene by PCR amplification and subsequent sequence analysis of the PCR fragments from 990 Chinese essential hypertensive subjects. We also made a comparative analysis of the collected data of the essential hypertension subjects who carried tRNALys mutation and those who did not carry the mutation using the methods of 1:1 case-control study. We totally found 7 mutation sites in 10 subjects. The onset ages of the individuals carrying the mutation were earlier than those who did not bear them. The level of blood urea nitrogen in hypertension subjects who carried tRNALys mutation was higher than the hypertension subjects who did not carried tRNALys mutation, while the serum potassium was significantly lower. The level of platelet count in hypertension subjects who carried tRNALys mutation was lower. The level of ventricular septal thickness in hypertension subjects who carried tRNALys mutation was higher and the level of left ventricular end diastolic diameter in hypertension subjects was significantly lower. Mitochondrial tRNALys mutations might result in the change of their structure and function, and then damaged the blood metabolism, the balance of the blood electrolyte, the steady-state of the blood cells and the heart structure and function, which were involved in the progress of the essential hypertension. Part of the essential hypertension patients clinically presented the characters of maternal inheritance, which might be associated with the tRNALys mutation. 相似文献
9.
10.
11.
12.
Goswami S Chatterjee S Bhattacharyya SN Basu S Adhya S 《Nucleic acids research》2003,31(19):5552-5559
Import of nucleus-encoded tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania involves recognition of specific import signals by the membrane-bound import machinery. Multiple signals on different tRNA domains may be present, and further, importable RNAs interact positively (Type I) or negatively (Type II) with one another at the inner membrane in vitro. By co-transfection assays, it is shown here that tRNATyr (Type I) transiently stimulates the rate of entry of tRNAIle (Type II) into Leishmania mitochondria in transfected cells, and conversely, is inhibited by tRNAIle. Truncation and mutagenesis experiments led to the co-localization of the effector and import activities of tRNATyr to the D domain, and those of tRNAIle to the variable region–T domain (V-T region), indicating that both activities originate from a single RNA–receptor interaction. A third tRNA, human tRNALys, is imported into Leishmania mitochondria in vitro as well as in vivo. This tRNA has Type I and Type II motifs in the D domain and the V-T region, respectively, and shows both Type I and Type II effector activities. Such dual-type tRNAs may interact simultaneously with the Type I and Type II binding sites of the inner membrane import machinery. 相似文献
13.
J.Peter Gergen Joel Y. Loewenberg Pieter C. Wensink 《Journal of molecular biology》1981,147(4):475-499
We have isolated segments of Drosophila melanogaster DNA that contain two clusters of tRNA2Lys genes. In one segment, pPW511, there is a cluster of three of these genes surrounded by other tRNA genes. Two other segments, pPW516 and pPW541. share a 3 × 103 base-pair region that has a cluster of four tRNA2Lys genes. This cluster is flanked by 20 × 103 base-pairs of DNA that does not appear to have other tRNA genes. The tRNA genes in both clusters are irregularly spaced and are intermingled with moderately repetitive DNA. Each cluster is present once or perhaps twice in the haploid genome and has the same arrangement of restriction endonuclease sites in the genomic DNA as in the isolated, cloned DNA. In situ hybridization to polytene chromosomes localized the pPW511 cluster to the 42A region and the pPW516/541 cluster to the 42E region. Another region, 50B, also contains tRNA2Lys genes. In sum, these cloned tRNA2Lys genes account for most of this gene family and are irregularly spaced in two clusters. 相似文献
14.
15.
16.
Daniel Klaiman Emmanuelle Steinfels-Kohn Gabriel Kaufmann 《Nucleic acids research》2014,42(1):328-339
Double-stranded DNA breaks (DSB) cause bacteria to augment expression of DNA repair and various stress response proteins. A puzzling exception educes the anticodon nuclease (ACNase) RloC, which resembles the DSB responder Rad50 and the antiviral, translation-disabling ACNase PrrC. While PrrC''s ACNase is regulated by a DNA restriction-modification (R-M) protein and a phage anti-DNA restriction peptide, RloC has an internal ACNase switch comprising a putative DSB sensor and coupled ATPase. Further exploration of RloC''s controls revealed, first, that its ACNase is stabilized by the activating DNA and hydrolysed nucleotide. Second, DSB inducers activated RloC''s ACNase in heterologous contexts as well as in a natural host, even when R-M deficient. Third, the DSB-induced activation of the indigenous RloC led to partial and temporary disruption of tRNAGlu and tRNAGln. Lastly, accumulation of CRISPR-derived RNA that occurred in parallel raises the possibility that the adaptive immunity and RloC provide the genotoxicated host with complementary protection from impending infections. 相似文献
17.
18.
An enzyme capable of liberating functional tRNALys from Escherichia coli diacetyl-lysyl-tRNALys was purified from the archae Sulfolobus solfataricus. Contrasting with the specificity of peptidyl- tRNA hydrolase (PTH) from E.coli, the S.solfataricus enzyme readily accepts E.coli formyl-methionyl-tRNAfMet as a substrate. N-terminal sequencing of this enzyme identifies a gene that has homologs in the whole archaeal kingdom. Involvement of this gene (SS00175) in the recycling of peptidyl-tRNA is supported by its capacity to complement an E.coli strain lacking PTH activity. The archaeal gene, the product of which appears markedly different from bacterial PTHs, also has homologs in all the available eukaryal genomes. Since most of the eukaryotes already display a bacterial-like PTH gene, this observation suggests the occurrence in many eukaryotes of two distinct PTH activities, either of a bacterial or of an archaeal type. Indeed, the bacterial- and archaeal-like genes encoding the two full-length PTHs of Saccharomyces cerevisiae, YHR189w and YBL057c, respectively, can each rescue the growth of an E.coli strain lacking endogeneous PTH. In vitro assays confirm that the two enzymes ensure the recycling of tRNALys from diacetyl-lysyl-tRNALys. Finally, the growth of yeast cells in which either YHR189w or YBL057c has been disrupted was compared under various culture conditions. Evidence is presented that YHR189w, the gene encoding a bacterial-like PTH, should be involved in mitochondrial function. 相似文献
19.
The yeast MTO1 gene encodes an evolutionarily conserved protein for the biosynthesis of the 5-carboxymethylaminomethyl group of cmnm5s2U in the wobble position of mitochondrial tRNA. However, mto1 null mutant expressed the respiratory deficient phenotype only when coupled with the C1409G mutation of mitochondrial 15S rRNA. To further understand the role of MTO1 in mitochondrial RNA metabolism, the yeast mto1 null mutants carrying either wild-type (PS) or 15S rRNA C1409G allele (PR) have been characterized by examining the steady-state levels, aminoacylation capacity of mitochondrial tRNA, mitochondrial gene expression and petite formation. The steady-state levels of tRNALys, tRNAGlu, tRNAGln, tRNALeu, tRNAGly, tRNAArg and tRNAPhe were decreased significantly while those of tRNAMet and tRNAHis were not affected in the mto1 strains carrying the PS allele. Strikingly, the combination of the mto1 and C1409G mutations gave rise to the synthetic phenotype for some of the tRNAs, especially in tRNALys, tRNAMet and tRNAPhe. Furthermore, the mto1 strains exhibited a marked reduction in the aminoacylation levels of mitochondrial tRNALys, tRNALeu, tRNAArg but almost no effect in those of tRNAHis. In addition, the steady-state levels of mitochondrial COX1, COX2, COX3, ATP6 and ATP9 mRNA were markedly decreased in mto1 strains. These data strongly indicate that unmodified tRNA caused by the deletion of MTO1 gene caused the instability of mitochondrial tRNAs and mRNAs and an impairment of aminoacylation of mitochondrial tRNAs. Consequently, the deletion of MTO1 gene acts in synergy with the 15S rRNA C1409G mutation, leading to the loss of COX1 synthesis and subsequent respiratory deficient phenotype. 相似文献
20.
Kirk Hayenga Charles Hedgcoth Mary Harrison Victor K. Lin B. J. Ortwerth 《Molecular and cellular biochemistry》1986,71(1):25-30
Summary Mouse lymphoma cells have three major isoaccepting lysine tRNAs. Two of these isoacceptors, tRNA2
Lys and tRNA4
Lys, were sequenced by rapid gel or chromatogram readout methods. They have the same primary sequence but differ in two modified nucleotides. tRNA4
Lys has an unmodified uridine replacing one dihydrouridine and an unidentified nucleotide, t6A*, replacing t6A. This unidentified nucleotide is not a hypomodified form of t6A. Thus, tRNA4ys is not a simple precursor of tRNA2
Lys. Both tRNAs have an unidentified nucleotide, U**, in the third position of the anticodon. Also, partial sequences of minor homologs of tRNA2
Lys and tRNA4
Lys were obtained. The distinctions between tRNA2
Lys and tRNA4
Lys may be part of significant cellular roles as illustrated by the differential effects of these isoacceptors on the synthesis by lysyl-tRNA synthetase of diadenosine-5,5-P1,P4-tetraphosphate, a putative signal in DNA replication. 相似文献