Exoribonucleases in wild type Escherichia coli and RNase II-deficient mutants.
Explore the source record for details and available documents.
Escherichia coli↗
SEARCH · PubMed Health
Results for “Exoribonucleases”
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
A structural bridge between dengue virus tandem xrRNAs facilitates coordination of exonuclease resistance.
Orthoflavivirus RNA genomes resist host 5'-3' exoribonucleases to produce subgenomic flaviviral RNAs (sfRNAs). This resistance is conferred by exoribonuclease-resistant RNA (xrRNA) structures within the viral 3' untranslated region that often occur in tandem, and whose function can be coupled. In dengue virus serotype 2 (DENV2), this coupling results in changing patterns of sfRNA identity and abundance associated with the ability of the virus to adapt to host vs. vector infections. The physical basis of this coupling was unknown. Using a combination of virology, biochemistry, bioinformatics, structural biology, and biophysics, we explored the structural and sequence determinants of tandem xrRNA coupling in DENV2. We discovered that the spatial proximity, order, and structural integrity of the tandem xrRNAs are all important for coupling. Furthermore, an unpaired A-rich linker that lies between the two xrRNAs is essential in stabilizing a specific structure that correlates to coupling. This A-rich sequence likely forms tertiary contacts with an adjacent stem-loop structure to form a physical bridge between the two xrRNAs, a finding that is supported by a mid-resolution cryo-electron microscopy (cryo-EM) map of the DENV2 tandem xrRNAs. Disruption of the structure of this bridge by mutation changes the relative orientation or spacing between the tandem xrRNAs, which is correlated to their functional coupling. These findings help provide an explanation for the coupling between tandem xrRNAs, suggesting a new mechanistic hypothesis in which the two tandem xrRNAs can simultaneously encounter Xrn1.IMPORTANCEDengue virus (DENV) generates non-coding subgenomic flaviviral RNAs (sfRNAs) that affect several cellular pathways and are important for successful infection. These sfRNAs are formed by structured RNA elements in the viral genome called exoribonuclease-resistant RNAs (xrRNAs), which fold into a distinct three-dimensional topology to block degradation by host cell exoribonucleases and often occur in tandem. Specific patterns of sfRNAs made during infection are important for host vs. vector fitness, and in DENV2, this pattern depends on functional coupling between tandem xrRNAs. However, the source of this functional coupling was unknown. We determined that an unpaired A-rich linker between the tandem xrRNAs is necessary for creating a structural bridge between the tandem xrRNAs. This bridge appears to favor a specific orientation between the tandem xrRNAs that is correlated to coupling and therefore to the patterns and relative abundance of sfRNAs produced during infection.
Dengue Virus↗
Trypanosoma brucei: calcium-dependent endoribonuclease is associated with inhibitor protein.
T. brucei cytoplasmic calcium-dependent alkaline ribonuclease activity from DEAE-cellulose fractionation was separated into endoribonuclease and exoribonuclease activities by hydroxyapatite chromatography. T. brucei cytoplasmic extract markedly decreased the endoribonuclease activity, but slightly potentiated the activities of the exoribonuclease and bovine ribonuclease A. While the endoribonuclease was activated by trypsin, the exoribonuclease and bovine ribonuclease A were partially inactivated by trypsin. The endoribonuclease was activated by p-chloromercuribenzoate or N-ethylmaleimide; the exoribonuclease was not affected by these sulfhydryl group reagents. Free ribonuclease was separated from the latent endoribonuclease by 1 M NaCl-Sephadex G-100 gel filtration. The results demonstrate that T. brucei cytoplasm contains a latent endoribonuclease consisting of ribonuclease and inhibitor protein.
Animals↗
Properties of oncornavirus RNA-directed DNA polymerase, the RNA template, and the intracellular products formed early during infection and cell transformation.
We have investigated three aspects of RNA turmor virus replication and cell transformation: (1) the properties of the purified avian and mammalian viral RNA-directed DNA polumerase, (2) some characteristics of the viral 60-70S RNA genome, 30-40S RNA subunits and intracellular viral RNA species, and (3) the interaction of the viral DNA polymerase with its RNA template early during infection and cell transformation by the murine sarcoma-leukemia virus (MSV[MLV]). Avian myeloblastosis virus (AMV) contains two forms of RNA-directed DNA polymerase, alpha, consisting of a single polypeptide of molecular weight 65,000, and alphabeta, consisting of two polypeptides of molecular weights 65,000 and 105,000. The alpha and alphabeta forms of AMV DNA polymerase both possess RNase H activity that requires free end termini on the ribopolymer and can degrade the RNA of the RNA-DNA hybrid in the 3' to 5' and 5' to 3' directions. But, alpha and alphabeta possess a different mode of exoribonuclease activity. While alphabeta RNase H is a processive exoribonuclease that degrades the polynucleotide chain to a core residue before attacking a second chain, alpha RNase H is a random exoribonuclease that releases the polynucleotide after each scission. Highly purified Moloney-MSV(MLV) DNA polymerase has both RNase H activity and the ability to read viral 60-70S RNA. These activities comigrate through five different steps of purification and are present at levels comparable to those found in purified AMV DNA polymerase. The MSV(MLV) 60-70S RNA genome and 35S RNA subunits were shown by periodate oxidationtritiated borohydride reduction to contain adenosine as the major 3'-terminal nucleoside. Poly (A) segments were isolated from viral 60-70S and 35S RNA by treatment with RNase A or RNase T1 and purified by afinity chromatography and gel electrophoresis. Viral poly(A) was shown to be present at the 3' terminus as -G(C,U)A190AOH. The similar sequence reported for poly(A) present in mammalian mRNA suggests that similar mechanisma are involved in the transcription and processing of both cellular and viral DNA sequences. Within transformed cells replicating MSV(MLV), viral 35S and 20S RNA were found in membrane-bound polyribosomes, whereas only 35S RNA was detected in free polyribosomes. The origin and function of 20S RNA is unknown. The early events during rapid infection and cell transformation of mouse 3T6 cells by the Harvey strain of MSV(MLV) were studied. By both autoradiographic analysis and molecular hybridization, viral DNA synthesis was detected in the cytoplasm by 1 hour after infection, reached a maximum at 2 hours, and subsequently decreased. Cytological chase experiments produced evidence that cytoplasmic viral DNA was transported to the nucleus. In situ hybridization experiments using radioactive viral DNA product as a probe demonstrated the rapid association of viral DNA sequences with the chromocenters of interphase nuclei and with the centromeric heterochromatin regions of some chromosomes.
Avian Myeloblastosis Virus↗
Rho-dependent termination and RNase E-mediated cleavage: dual pathways for RNA 3' end processing in polycistronic mRNA.
"Pre-full-length" transcripts are produced at the end of the polycistronic galactose (gal) operon, 5' galE-galT-galK-galM 3', via Rho-dependent transcription termination (RDT) and -independent transcription termination. The 3' end of the full-length galETKM mRNA is acquired by exonucleolytic processing of the 3'-OH ends of the pre-full-length transcripts. However, the gal operon produces an mRNA termed galE whose 3' end forms approximately 120 nucleotides downstream of the galE stop codon, within the subsequent gene, galT, thereby establishing polarity in gene expression. In this study, we investigated the molecular processes that generate the 3' end of galE mRNA. We discovered that the 3' ends of pre-galE mRNA are produced in the middle of galT as a result of the combination of two separate molecular processes-one previously reported as RDT and the other as unreported RNase E-mediated transcript cleavage. The 3' ends of pre-galE mRNA undergo exonucleolytic processing to the 3' end of galE mRNA observed in vivo. A hairpin structure containing an 8 bp stem and a 4-nucleotide loop, located 5-10 nucleotides upstream of the 3' ends of galE mRNA, blocks exoribonuclease digestion and renders transcript stability. These findings demonstrate that RNase E-contrary to its general role in mRNA degradation-produces RNA 3' ends that regulate polarity in gene expression.IMPORTANCEThis study reports the findings of two molecular mechanisms that generate the 3' ends of pre-galE mRNA in the gal operon, viz., Rho-dependent transcription termination and RNase E-mediated cleavage. These 3' ends are subsequently processed to produce stable galE mRNA with a hairpin structure that prevents exoribonuclease degradation. This mechanism establishes gene expression polarity by generating the 3' end of galE mRNA within galT in contrast to the usual mRNA degradation role of RNase E. The study reveals a unique role of RNase E in mRNA processing and stability.
RNA, Messenger↗
Alterations of activities of ribonucleases and polyadenylate polymerase in synchronized mouse L cells.
The activities of the three known catabolic and the one anabolic polyadenylate enzymes have been determined in synchronized L5178y cells: endoribonuclease, exoribonuclease, 5'-nucleotidase and poly(A) polymerase (Mg2+-dependent). These four enzymes were found primarily in the nuclear fraction. The activity of poly(A) polymerase remains essentially constant during the transition from G1 to S phase. However, the poly(A) catabolic enzyme activities increase parallel with DNA synthesis; the endoribonuclease activity increases 4-fold during G1 to S phase, the exoribonuclease and the nucleotidase activities increasing 30-fold and 16-fold. During the S phase the poly(A)-degrading enzymes are far more active than the poly(A)-synthesizing activity of poly(A) polymerase. We conclude that in L5178y cells the poly(A)-degrading enzymes probably function in regulation of the post-transcriptional net-polyadenylation of heterogeneous nuclear RNA during the phase of DNA synthesis.
Cell Division↗
Age-dependent enzymatic poly (A) metabolism in quail oviduct.
The poly(A) metabolism in oviducts from adult and senescent quails has been studied. The incorporation studies by double-labelling of mRNA with [3H]adenosine and [3H]uridine revealed, that after inhibition of transcription by actinomycin D, the incorporation ratio adenosine/uridine increases drastically in adult animals compared with the ratio determined for mRNA from senescent animals. This finding is a hint that in senescent animals the poly(A) stretch of mRNA is shorter than in adult animals. This assumption is supported by the finding that the activity of the extractable poly(A) exoribonuclease is higher in senescent than in adult animals. The activity of poly(A) exoribonuclease is even enhanced after stimulation of senescent animals with progesterone. The activity of the other catabolic enzyme studied, the poly (A) endoribonuclease, as well as the activity of anabolic poly(A) enzyme, the poly(A) polymerase, are almost identical in oviducts from adult and senescent animals. The properties of the catabolic poly(A) enzymes and of the anabolic poly(A) polymerase seem to be identical.
Adenosine↗
Transcriptome-Wide Analysis of the 5' Cap Status of RNA Using 5' Monophosphate-Dependent Exonuclease Digestion and RNA Sequencing.
Eukaryotic mRNAs carry an N7-methylguanosine (m7G) cap structure at their 5' extremity, which protects them from the degradation by 5'-3' exoribonucleases and plays a pivotal role in mRNA metabolism, promoting splicing, nuclear export, and translation. Decapping, the enzymatic process that removes this structure, is a key event during cytoplasmic mRNA 5'-3' decay, leading to the degradation of the transcript body by Xrn1. In this chapter, we describe a procedure to assess the cap status of RNA at the transcriptome level. It is based on a treatment of total RNA extracts with a 5' monophosphate-dependent exonuclease, which like Xrn1 specifically degrades decapped RNAs harboring 5' monophosphate extremities, but not RNAs with intact m7G cap. The digested RNAs are then analyzed by RNA sequencing.
Exoribonucleases↗
Chloroplast mRNA 3' end maturation is biochemically distinct from prokaryotic mRNA processing.
We have characterized a chloroplast processing activity that catalyzes the conversion of the plastid cytochrome b6/f subunit IV (pet D) mRNA 3' end precursor to the mature RNA possessing a 3' inverted repeat (IR). In a chloroplast soluble protein extract, the activity requires Mg2+ or Mn2+, but not K+. In the absence of Mg2+, the pet D 3' IR-RNA product does not accumulate, and UV-cross-linking indicates that the 3' IR-RNA precursor binds several new proteins in addition to those previously characterized as part of the 3' IR-RNA: protein complex in vitro. In contrast, high concentrations of Zn2+ or Cu2+ suppress protein binding and inhibit the processing reaction. The purified exoribonuclease polynucleotide phosphorylase (E.C.2.7.7.8) is not efficient in processing the pet D 3' IR-RNA precursor, whereas Escherichia coli ribonuclease II rapidly processes the pet D IR-RNA precursor to a product of a size similar to that of the mature 3' IR-RNA, but also rapidly degrades the mature RNA in the absence of chloroplast extract. We therefore conclude that the maturation of the pet D mRNA in vitro requires specific chloroplast enzymes which process the mRNA 3' end precursor in the absence of efficient transcription termination. The chloroplast enzyme activities are biochemically distinct from their bacterial counterparts. We also note that specific chloroplast components may be required to stabilize the mature pet D mRNA 3' end against further exonucleolytic degradation.
Bacteria↗
RNase PH catalyzes a synthetic reaction, the addition of nucleotides to the 3' end of RNA.
Escherichia coli RNase PH is a phosphate-dependent exoribonuclease that has been implicated in the 3' processing of tRNA precursors. It degrades RNA chains in a phosphorolytic manner releasing nucleoside diphosphates as products. Here we show that RNase PH also catalyzes a synthetic reaction, the addition of nucleotides to the 3' termini of RNA molecules. The synthetic activity co-purifies with RNase PH throughout an extensive enrichment indicating that it is due to the same enzyme. The synthetic activity can incorporate all nucleoside diphosphates, but not triphosphates, and is strongly inhibited by Pi, but not PPi. Various RNA molecules stimulate nucleotide incorporation, and with tRNA the 3' end of the molecule serves a primer function. RNA chains as long as 40 residues can be synthesized in this system. As with polynucleotide phosphorylase, the synthetic activity of RNase PH apparently represents the reversal of the degradative reaction.
Exoribonucleases↗
RNase PH: an Escherichia coli phosphate-dependent nuclease distinct from polynucleotide phosphorylase.
Final trimming of the 3' terminus of tRNA precursors in Escherichia coli is thought to proceed by an exonucleolytic mechanism. However, mutant strains lacking as many as four exoribonucleases known to act on tRNA still grow normally and process tRNA normally. Extracts from such a multiple-RNase-deficient strain accurately mature tRNA precursors exonucleolytically in vitro in a reaction that requires inorganic phosphate. Here we show that this reaction is not due to polynucleotide phosphorylase (PNPase) but, rather, that it is mediated by a phosphate-requiring exonuclease that we have named RNase PH. Purified PNPase is incapable of completely processing tRNA precursors, and extracts from a PNPase- strain retain full activity for phosphorolytic processing. Although both PNPase and RNase PH act in a phosphorolytic manner, they differ substantially in size and substrate specificity. RNase PH has a molecular mass of 45-50 kDa and favors tRNA precursors as substrates. The possible physiological role of RNase PH and the advantages of phosphorolytic processing are discussed.
Bacterial Proteins↗
Analysis of spliceosome assembly and the structure of a regulated intron in Drosophila in vitro splicing extracts.
We characterize spliceosome assembly in Drosophila embryonic nuclear extracts. Further, we show that these extracts contain high levels of a 5' to 3' exoribonuclease activity allowing rapid, convenient protection mapping of 5' splice site and branchpoint sequences. We use this assay to show, for the first time, that a regulated arthropod intron uses a remote branchpoint strikingly similar in structure to those observed previously in regulated vertebrate introns. These results provide new evidence that both regulated and constitutive splicing are similar in detail in vertebrates and arthropods indicating that the powerful genetic systems for analysis of splicing regulation in Drosophila are likely to be directly informative for regulated splicing throughout metazoa. In addition, we report formation of a novel class of intron-dependent complexes. Behavior of these complexes indicates that they represent a mutually exclusive, kinetically competing pathway with spliceosome assembly. We propose that this competition represents the basis for a kinetic proofreading mechanism enhancing fidelity of intron recognition. We also discuss possible implications of this model for regulated splicing.
Animals↗
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.
Blotting, Southern↗
Identification of the rph (RNase PH) gene of Bacillus subtilis: evidence for suppression of cold-sensitive mutations in Escherichia coli.
A shotgun cloning of Bacillus subtilis DNA into pBR322 yielded a 2-kb fragment that suppresses the cold-sensitive defect of the nusA10(Cs) Escherichia coli mutant. The responsible gene encodes an open reading frame that is greater than 50% identical at the amino acid level to the E. coli rph gene, which was formerly called orfE. This B. subtilis gene is located at 251 degrees adjacent to the gerM gene on the B. subtilis genetic map. It has been named rph because, like its E. coli analog, it encodes a phosphate-dependent exoribonuclease activity, RNase PH, that removes the 3' nucleotides from precursor tRNAs. The cloned B. subtilis rph gene also suppresses the cold-sensitive phenotype of other unrelated cold-sensitive mutants of E. coli, but not the temperature-sensitive phenotype of three temperature-sensitive mutants, including the nusA11(Ts) mutant, that were tested.
Amino Acid Sequence↗
Proper 5'-3' cotranslational mRNA decay in yeast requires import of Xrn1 to the nucleus.
The budding yeast Xrn1 protein shuttles between the nucleus, where it stimulates transcription, and the cytoplasm, where it executes the major cytoplasmic mRNA decay. In the cytoplasm, apart from catalyzing 5'→3' decay onto non translated mRNAs, Xrn1 can follow the last translating ribosome to degrade the decapped mRNA template, a process known as "cotranslational mRNA decay". We have previously observed that the import of Xrn1 to the nucleus is required for efficient cytoplasmic mRNA decay. Here by using an Xrn1 mutant that cannot enter the nucleus, but is otherwise functional in ribonuclease activity, we show that nuclear import is necessary for proper global cotranslational decay of mRNAs along coding regions and also affects degradation in the of 5' region of a large group of mRNAs, which comprise about 20% of the transcriptome. Furthermore, a principal component analysis of the genomic datasets of this mutant and other Xrn1 mutants also shows that lack of a cytoplasmic 5'→3' exoribonuclease is the primary cause of the physiological defects seen in a xrn1Δ mutant, but also suggests that Xrn1 import into the nucleus is necessary for its full in vivo functions.
Exoribonucleases↗
Sequence and transcriptional analysis of the Escherichia coli rnt gene encoding RNase T.
The Escherichia coli rnt gene encoding the enzyme RNase T, which is responsible for the end-turnover of tRNA, was cloned on a 1.5-kilobase DNA fragment. When placed in pUC18 and pUC19 vectors this fragment led to approximately a 40-fold overexpression of RNase T activity. The cloned fragment was sequenced and was found to contain an open reading frame sufficient to encode a protein of 215 amino acids with a molecular weight of 23,521, which is close to the subunit molecular weight of RNase T; the fragment also contains a second incomplete open reading frame with some sequence similarity to RNA helicases. The derived sequence of RNase T showed no similarity to any of the other E. coli exoribonucleases sequenced to date. Primer extension analysis and deletion of part of the upstream region were used to identify the transcription start point and the promoter of the rnt gene. Northern and primer extension analysis revealed that the rnt message also included the second open reading frame, suggesting that rnt is part of an operon.
Amino Acid Sequence↗
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.
Escherichia coli↗
The unusual stability of the IS10 anti-sense RNA is critical for its function and is determined by the structure of its stem-domain.
IS10 transposition is regulated by an approximately 70 nt anti-sense RNA, RNA-OUT. RNA-OUT folds into a duplex 'stem-domain' topped by a loosely paired 'loop-domain'. The loop-domain is critical for RNA-RNA pairing per se; pairing initiates by interaction of the RNA-OUT loop with the 5' end of the target mRNA. We show here that RNA-OUT is unusually stable in vivo (half-life 60 min) and that this stability is conferred by specific features of the RNA-OUT stem-domain. One critical feature is stable base-pairing: mutations that disrupt stem pairing destabilize RNA-OUT in vivo and abolish anti-sense control; combinations of mutations that restore pairing also restore both stability and control. We propose that the stem renders RNA-OUT resistant to 3' exoribonucleases. Other features of the stem-domain prevent this essential duplex from being an effective substrate for double-strand nucleases: two single base mutations disrupt antisense control by making RNA-OUT susceptible to RNase III. Mutations in the loop region have little effect on RNA-OUT stability. Implications for IS10 biology and the design of efficient anti-sense RNAs are discussed.
Bacteriophage lambda↗