Transfer RNA is a substrate for RNase D in vivo

RNase D is a 3'-exonuclease whose in vitro specificity has suggested a role in tRNA processing. However, since mutant Escherichia coli strains devoid of RNase D display a normal phenotype, it has not been possible to ascertain the enzyme's function or even to determine which RNA is its substrate in vivo. Here we show that transformation of strains devoid of tRNA nucleotidyltransferase with a multicopy plasmid carrying the rnd+ gene leads to extremely slow growth due to elevated levels of RNase D activity. Analysis of such a slow-growing strain revealed that less tRNA is present in the cell and that the tRNA that could be recovered is substantially damaged. These studies demonstrate that RNase D can act at the 3' terminus of tRNA in vivo, and they support the conclusion that RNase D participates in tRNA metabolism.     

Escherichia coli RNase D: sequencing of the rnd structural gene and purification of the overexpressed protein.

We have determined the nucleotide sequence of a 1.4-kb-pair fragment of the E. coli chromosome that carries the complete rnd gene encoding RNase D, a putative tRNA processing enzyme. The coding region of rnd extends for a total of 1128 nucleotides beginning at an initiator UUG codon and terminating at a UAA codon, and encodes a 375-amino acid polypeptide of 42,679 daltons, consistent with the known size of RNase D. A rapid purification procedure was developed for isolation of RNase D from strains overexpressing the enzyme. The N-terminal sequence and the amino acid composition of the homogenous protein were in excellent agreement with those derived from the sequence of the rnd gene.     

Ribonuclease D is not essential for the normal growth of Escherichia coli or bacteriophage T4 or for the biosynthesis of a T4 suppressor tRNA

Transposon Tn10-mediated rearrangement was used to isolate a strain of Escherichia coli carrying a deletion in the rnd region which is known to encode the structural gene for the putative 3' tRNA processing nuclease, RNase D. Genetic analysis indicated that about 0.4-0.5 min of the chromosome in the 39.5-40.0 min region was deleted. The mutant strain was devoid of RNase D activity, but other RNase activities were unaffected. The viability of the mutant strain and its normal growth characteristics indicate that RNase D is not essential for E. coli survival. The normal plating efficiency in this mutant host of wild type T4 and a T4 psu1+-amber double mutant indicates that RNase D is also not required for T4 growth or psu1+-tRNA processing. The implications of these findings for the role of RNase D in bacterial and bacteriophage tRNA metabolism, and the possible involvement of alternative enzymes, are discussed.     

The Agrobacterium tumefaciens rnd homolog is required for TraR-mediated quorum-dependent activation of Ti plasmid tra gene expression

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via TraR and its cognate autoinducer, N-(3-oxo-octanoyl)-L-homoserine lactone. We isolated four Tn5-induced mutants of A. tumefaciens C58 deficient in TraR-mediated activation of tra genes on pTiC58DeltaaccR. These mutations also affected the growth of the bacterium but had no detectable influence on the expression of two tester gene systems that are not regulated by quorum sensing. In all four mutants Tn5 was inserted in a chromosomal open reading frame (ORF) coding for a product showing high similarity to RNase D, coded for by rnd of Escherichia coli, an RNase known to be involved in tRNA processing. The wild-type allele of the rnd homolog cloned from C58 restored the two phenotypes to each mutant. Several ORFs, including a homolog of cya2, surround A. tumefaciens rnd, but none of these genes exerted a detectable effect on the expression of the tra reporter. In the mutant, traR was expressed from the Ti plasmid at a level about twofold lower than that in NT1. The expression of tra, but not the growth rate, was partially restored by increasing the copy number of traR or by disrupting traM, a Ti plasmid gene coding for an antiactivator specific for TraR. The mutation in rnd also slightly reduced expression of two tested vir genes but had no detectable effect on tumor induction by this mutant. Our data suggest that the defect in tra gene induction in the mutants results from lowered levels of TraR. In turn, production of sufficient amounts of TraR apparently is sensitive to a cellular function requiring RNase D.     

RNase T affects Escherichia coli growth and recovery from metabolic stress.

To determine the essentiality and role of RNase T in RNA metabolism, we constructed an Escherichia coli chromosomal rnt::kan mutation by using gene replacement with a disrupted, plasmid-borne copy of the rnt gene. Cell extracts of a strain with mutations in RNases BN, D, II, and I and an interuppted rnt gene were devoid of RNase T activity, although they retained a low level (less than 10%) of exonucleolytic activity on tRNA-C-C-[14C]A due to two other unidentified RNases. A mutant lacking tRNA nucleotidyltransferase in addition to the aforementioned RNases accumulated only about 5% as much defective tRNA as did RNase T-positive cells, indicating that this RNase is responsible for essentially all tRNA end turnover in E. coli. tRNA from rnt::kan strains displayed a slightly reduced capacity to be aminoacylated, raising the possibility that RNase T may also participate in tRNA processing. Strains devoid of RNase T displayed slower growth rates than did the wild type, and this phenotype was accentuated by the absence of the other exoribonucleases. A strain lacking RNase T and other RNases displayed a normal response to UV irradiation and to the growth of bacteriophages but was severely affected in its ability to recover from a starvation regimen. The data demonstrate that the absence of RNase T affects the normal functioning of E. coli, but it can be compensated for to some degree by the presence of other RNases. Possible roles of RNase T in RNA metabolism are discussed.     

RNase PH is essential for tRNA processing and viability in RNase-deficient Escherichia coli cells.

RNase PH is a Pi-dependent exoribonuclease that can act at the 3' terminus of tRNA precursors in vitro. To obtain information about the function of this enzyme in vivo, the Escherichia coli rph gene encoding RNase PH was interrupted with either a kanamycin resistance or a chloramphenicol resistance cassette and transferred to the chromosome of a variety of RNase-resistant strains. Inactivation of the chromosomal copy of rph eliminated RNase PH activity from extracts and also slowed the growth of many of the strains, particularly ones that already were deficient in RNase T or polynucleotide phosphorylase. Introduction of the rph mutation into a strain already lacking RNases I, II, D, BN, and T resulted in inviability. The rph mutation also had dramatic effects on tRNA metabolism. Using an in vivo suppressor assay we found that elimination of RNase PH greatly decreased the level of su3+ activity in cells deficient in certain of the other RNases. Moreover, in an in vitro tRNA processing system the defect caused by elimination of RNase PH was shown to be the accumulation of a precursor that contained 4-6 additional 3' nucleotides following the -CCA sequence. These data indicate that RNase PH can be an essential enzyme for the processing of tRNA precursors.     

tRNA nucleotidyltransferase is not essential for Escherichia coli viability.

The role of tRNA nucleotidyltransferase in Escherichia coli has been uncertain because all tRNA genes studied in this organism already encode the -C-C-A sequence. Examination of a cca mutant, originally thought to contain 1-2% enzyme activity, indicated that it actually produces an inactive fragment of 40 kd compared to 47 kd for the wild-type enzyme due to a nonsense mutation in its cca gene. To confirm that the residual activity in extracts of this strain is due to another enzyme, and that tRNA nucleotidyltransferase is non-essential, we have interrupted the cca gene in vitro, and transferred this mutant gene to a variety of strains. In all cases mutant strains are viable, although as much as 15% of the tRNA population contains defective 3' termini, and no tRNA nucleotidyltransferase is detectable. Mutant strains grow slowly, but can be restored to more normal growth by a relA mutation or by a decrease in RNase T activity. In the latter case the amount of defective tRNA decreases dramatically. These findings indicate that tRNA nucleotidyltransferase is not essential for E. coli viability, and therefore, that all essential tRNA genes in this organism encode the -C-C-A sequence.     

Bacillus subtilis RNase III gene: cloning, function of the gene in Escherichia coli, and construction of Bacillus subtilis strains with altered rnc loci.

The rnc gene of Bacillus subtilis, which has 36% amino acid identity with the gene that encodes Escherichia coli RNase III endonuclease, was cloned in E. coli and shown by functional assays to encode B. subtilis RNase III (Bs-RNase III). The cloned B. subtilis rnc gene could complement an E. coli rnc strain that is deficient in rRNA processing, suggesting that Bs-RNase III is involved in rRNA processing in B. subtilis. Attempts to construct a B. subtilis rnc null mutant were unsuccessful, but a strain was constructed in which only a carboxy-terminal truncated version of Bs-RNase III was expressed. The truncated Bs-RNase III showed virtually no activity in vitro but was active in vivo. Analysis of expression of a copy of the rnc gene integrated at the amy locus and transcribed from a p(spac) promoter suggested that expression of the B. subtilis rnc is under regulatory control.     

Escherichia coli rna gene encoding RNase I: cloning, overexpression, subcellular distribution of the enzyme, and use of an rna deletion to identify additional RNases.

The cloning and overexpression of the Escherichia coli rna gene encoding RNase I are described. Only a single copy of the rna gene is present on the E. coli chromosome. Although cells with as much as a 100-fold increase in RNase I activity were constructed, little effect on cell growth was observed. Overexpressed RNase I was found in the periplasmic space to the same degree (approximately 85%) as wild-type enzyme, suggesting no limitation in RNase I transport. The rna clone was used to identify a deletion strain totally lacking the rna gene. The normal growth of this strain showed that RNase I is not essential for cell viability. Extracts from the RNase I deletion strain still retained a low level of RNase activity in the presence of EDTA, conclusively demonstrating the existence of additional EDTA-active RNases in E. coli. The possibility of a RNase I inhibitor is also discussed.     

A single base change in the acceptor stem of tRNA(3Leu) confers resistance upon Escherichia coli to the calmodulin inhibitor, 48/80

We have isolated several classes of spontaneous mutants resistant to the calmodulin inhibitor 48/80 which inhibits cell division in Escherichia coli K12. Several mutants were also temperature sensitive for growth and this property was exploited to clone a DNA fragment from an E. coli gene library restoring growth at 42 degrees C and drug sensitivity at 30 degrees C in one such mutant. Physical and genetic mapping confirmed that both the mutation and the cloned DNA were located at 15.5 min on the E. coli chromosome at a locus designated feeB. By subcloning, complementation analysis and sequencing, the feeB locus was identified as identical to the tRNA(CUALEU) gene. When the mutant locus was isolated and sequenced, the mutation was confirmed as a single base change, C to A, at position 77 in the acceptor stem of this rare Leu tRNA. In other studies we obtained evidence that this mutant tRNA, recognizing the rare Leu codon, CUA, was defective in translation at both permissive and non-permissive temperatures. The feeB1 mutant is defective in division and shows a reduced growth rate at non-permissive temperature. We discuss the possibility that the mutant tRNA(3Leu) is limiting for the synthesis of a polypeptide(s), requiring several CUA codons for translation which in turn regulates in some way the level or activity of the drug target, a putative cell cycle protein.     

Molecular cloning of the cls gene responsible for cardiolipin synthesis in Escherichia coli and phenotypic consequences of its amplification.

The cls gene responsible for cardiolipin synthesis in Escherichia coli K-12 was cloned in a 5-kilobase-pair DNA fragment inserted in a mini-F vector, pML31, and then subcloned into a 2.0-kilobase-pair fragment inserted in pBR322. The initial selection of the gene was accomplished in a cls pss-1 double mutant that had lesions in both cardiolipin and phosphatidylserine synthases and required either the cls or the pss gene product for normal growth at 42 degrees C in a broth medium, NBY, supplemented with 200 mM sucrose. The cloned gene was identified as the cls gene by the recovery and amplification of both cardiolipin and cardiolipin synthase in a cls mutant as well as by the integration of a pBR322 derivative into its genetic locus at 27 min on the chromosome of a polA1 mutant. The maxicell analysis indicated that a protein of molecular weight 46,000 is the gene product. The cls gene is thus most likely the structural gene coding for cardiolipin synthase. Hybrid plasmids of high copy numbers containing the cls gene were growth inhibitory to pss-I mutants under the above selective conditions, whereas they inhibited neither the growth of pss-I mutants at 30 degrees C nor that of pss+ strains at any temperature. Amplification of cardiolipin synthase activity was observed, but was not proportional to the probable gene dosage (the enzyme activity was at most 10 times that in wild-type cells), and cardiolipin synthesis in vivo was at the maximum 1.5 times that in wild-type strains, implying the presence in E. coli cells of a mechanism that avoids cardiolipin overproduction, which is possibly disadvantageous to proper membrane functions.     

Cloning, sequencing, and species relatedness of the Escherichia coli cca gene encoding the enzyme tRNA nucleotidyltransferase

The Escherichia coli cca gene which encodes the enzyme tRNA nucleotidyltransferase has been cloned by taking advantage of its proximity to the previously cloned dnaG locus. A series of recombinant bacteriophages, spanning the chromosomal region between the dnaG and cca genes at 66 min on the E. coli linkage map, were isolated from a lambda Charon 28 partial Sau3A E. coli DNA library using recombinant plasmids containing regions between dnaG and cca as probes. Two of the recombinant phage isolates, lambda c1 and lambda c4, contained the cca gene. A BamHI fragment from lambda c1 was subcloned into pBR328, and cells containing this recombinant plasmid, pRH9, expressed tRNA nucleotidyltransferase activity at about 10-fold higher level than the wild type control. The cca gene was further localized to a 1.4-kilobase stretch of DNA by Bal31 deletion analysis. The nucleotide sequence of the cca gene was determined by the dideoxy method, and revealed an open reading frame extending for a total of 412 codons from an initiator GTG codon that would encode a protein of about 47,000 daltons. Southern analysis using genomic blots demonstrated that the cca gene is present as a single copy on the E. coli chromosome and that there is no homology on the DNA level between the E. coli cca gene, and the corresponding gene in the Bacillus subtilis, Saccharomyces cerevisiae, Petunia hybrida, or Homo sapiens genomes. Homology was found only with DNA from the closely related species, Salmonella typhimurium. These studies have also allowed exact placement of the cca gene on the E. coli genetic map, and have shown that it is transcribed in a clockwise direction.     

RPM2, independently of its mitochondrial RNase P function, suppresses an ISP42 mutant defective in mitochondrial import and is essential for normal growth.

RPM2 is identified here as a high-copy suppressor of isp42-3, a temperature-sensitive mutant allele of the mitochondrial protein import channel component, Isp42p. RPM2 already has an established role as a protein component of yeast mitochondrial RNase P, a ribonucleoprotein enzyme required for the 5' processing of mitochondrial precursor tRNAs. A relationship between mitochondrial tRNA processing and protein import is not readily apparent, and, indeed, the two functions can be separated. Truncation mutants lacking detectable RNase P activity still suppress the isp42-3 growth defect. Moreover, RPM2 is required for normal fermentative yeast growth, even though mitochondrial RNase P activity is not. The portion of RPM2 required for normal growth and suppression of isp42-3 is the same. We conclude that RPM2 is a multifunctional gene. We find Rpm2p to be a soluble protein of the mitochondrial matrix and discuss models to explain its suppression of isp42-3.     

Characterization of the RNA processing enzyme RNase III from wild type and overexpressing Escherichia coli cells in processing natural RNA substrates.

1. A precursor to small stable RNA, 10Sa RNA, accumulates in large amounts in a temperature sensitive RNase E mutant at non-permissive temperatures, and somewhat in an rnc (RNase III-) mutant, but not in an RNase P- mutant (rnp) or wild type E. coli cells. 2. Since p10Sa RNA was not processed by purified RNase E and III in customary assay conditions, we purified p10Sa RNA processing activity about 700-fold from wild type E. coli cells. 3. Processing of p10Sa RNA by this enzyme shows an absolute requirement for a divalent cation with a strong preference for Mn2+ over Mg2+. Other divalent cations could not replace Mn2+. 4. Monovalent cations (NH+4, Na+, K+) at a concentration of 20 mM stimulated the processing of p10Sa RNA and a temperature of 37 degrees C and pH range of 6.8-8.2 were found to be optimal. 5. The enzyme retained half of its p10Sa RNA processing activity after 30 min incubation at 50 degrees C. 6. Further characterization of this activity indicated that it is RNase III. 7. To further confirm that the p10Sa RNA processing activity is RNase III, we overexpressed the RNase III gene in an E. coli cells that lacks RNase III activity (rnc mutant) and RNase III was purified using one affinity column, agarose.poly(I).poly(C). 8. This RNase III preparation processed p10Sa RNA in a similar way as observed using the p10Sa RNA processing activity purified from wild type E. coli cells, confirming that the first step of p10Sa RNA processing is carried out by RNase III.