Metal ion requirements and other aspects of the reaction catalyzed by M1 RNA, the RNA subunit of ribonuclease P from Escherichia coli

M1 RNA, the RNA subunit of ribonuclease P from Escherichia coli, can under certain conditions catalytically cleave precursors to tRNA in the absence of C5, the protein moiety of RNase P. M1 RNA itself is not cleaved during the reaction, nor does it form any covalent bonds with its substrate. Only magnesium and, to a lesser extent, manganese ions can function at the catalytic center of M1 RNA. Several other ions either inhibit the binding of magnesium ion at the active site or function as structural counterions. The reaction rate of cleavage of precursors to tRNAs by M1 RNA is enhanced in the presence of poly-(ethylene glycol) or 2-methyl-2,4-pentanediol. Many aspects of the reaction catalyzed by M1 RNA are compatible with a mechanism in which phosphodiester bond cleavage is mediated by metal ion.     

A novel tertiary interaction in M1 RNA, the catalytic subunit of Escherichia coli RNase P.

Phylogenetic covariation of the nucleotides corresponding to the bases at positions 121 and 236 in Escherichia coli RNase P RNA (M1 RNA) has been demonstrated in eubacterial RNase P RNAs. To investigate whether the nucleotides at these positions interact in M1 RNA we introduced base substitutions at either or at both of these positions. Single base substitutions at 121 or at 236 resulted in M1 RNA molecules which did not complement the temperature-sensitive phenotype associated with rnpA49 in vivo whereas wild-type M1 RNA or the double mutant M1 RNA, with restored base-pairing between 121 and 236, did. In addition, wild-type and the double mutant M1 RNA were efficiently cleaved by Pb++ between positions 122 and 123 whereas the rate of this cleavage was significantly reduced for the singly mutated M1 RNA variants. From these data we conclude that the nucleotides at positions 121 and 236 in M1 RNA establish a novel long-range tertiary interaction in M1 RNA. Our results also demonstrated that this interaction is not absolutely required for cleavage in vitro, however, a disruption resulted in a reduction in cleavage efficiency (kcat/Km), both in the absence and presence of C5.     

Processing of RNA in Escherichia coli is limited in the absence of ribonuclease III,ribonuclease E and ribonuclease P

A strain of Escherichia coli lacking RNAase III and containing thermolabile RNAase E and RNAase P was labeled with ³²P_i at a non-permissive temperature. RNA molecules were separated by two-dimensional polyacrylamide gel electrophoresis. Most of the small RNA species were isolated and analyzed for the presence of 5′ nucleoside triphosphates. In 16 of the 22 species analyzed a significant number of the individual molecules contained 5′ di or triphosphates. We conclude, therefore, that very little endonucleolytic RNA processing occurs in the absence of the three RNA processing enzymes RNAase III, RNAase E and RNAase P.     

Cleavage of tRNA precursors by the RNA subunit of E. coli ribonuclease P (M1 RNA) is influenced by 3'-proximal CCA in the substrates

tRNA precursor molecules that contain the CCA sequence found at the 3' termini of all mature tRNAs are cleaved in vitro more readily by M1 RNA, the catalytic subunit of E. coli RNAase P, than precursors that lack this sequence. The sensitivity to the CCA sequence is not apparent when precursors are cleaved by the reconstituted RNAase P holoenzyme that contains both M1 RNA and the protein subunit. These results have been obtained with monomeric precursor molecules encoded by the E. coli and human chromosomes and with three dimeric precursor molecules encoded by the bacteriophage T4 genome. The data are in agreement with previous results concerning T4 tRNA biosynthesis in vivo and show that the CCA sequence is important for the processing of precursors to tRNAs.     

The P3 domain of E. coli ribonuclease P RNA can be truncated and replaced

We prepared some truncated and replaced P3 mutants of Escherichia coli RNase P RNA, and used them to examine the RNase P ribozyme and holoenzyme reactions of a pre-tRNA substrate. The results indicated that mutations in the P3 domain did not affect the cleavage site selection of the pre-tRNA substrate, but did affect the efficiency of cleavage of the substrate. Results of stepwise truncation of the P3 domain and its replacement by the TAR sequence showed that the P3 domain of the E. coli RNase P was able to be truncated to certain length and was replaceable, but could not be deleted in the ribozyme.     

Properties of purified ribonuclease P from Escherichia coli

The purified protein moiety of ribonuclease P (EC 3.1.26.5) from Escherichia coli, a single polypeptide of molecular weight approximately 17 500, has not catalytic activity by itself on several RNA substrates. However, when it is marked in vitro with an RNA species called M1 RNA, RNase P activity is reconstituted. The rate at which the purified RNase P cleaves any particular tRNA precursor molecule depends on the identity of that tRNA precursor.     

Methionyl-tRNA synthetase-induced conformational change of Escherichia coli tRNAfMet

Ribonuclease T2, nuclease S1, and snake venom phosphodiesterase were used as a structural probe for investigation of the interaction between Escherichia coli tRNAfMet and methionyl-tRNA synthetase, and the cleavage sites were analyzed by a rapid sequencing gel electrophoresis of 5'-32P-labeled tRNA. Both endonucleases cleaved the D-loop of synthetase-bound tRNA much more extensively than that of the free tRNA. Positions of A14, G15, A22, and G23 in the D-loop and C35 in the anticodon of the synthetase-bound tRNA were more susceptible to RNase T2. The synthetase-bound tRNA was predominantly cleaved by nuclease S1 at position of G15, G19, G20, and G23 in the D-loop and G2 in the acceptor stem. In contrast, the synthetase-bound tRNA was more resistant to the 3'-exonuclease, snake venom phosphodiesterase, than was the free tRNA molecule. These results suggest conformational change of the tRNA by the synthetase binding which weakened tertiary interaction between the D-loop and T psi C-loop/extra-loop. Production of acid-soluble radioactivity was also examined in the limited digestion of 5'-32P-labeled tRNA or 3'-14C-labeled methionyl-tRNA. The synthetase enhanced the release of acid-soluble oligonucleotides from the 5'-end of the tRNA but suppressed that from the 3'-end of the molecule. These results are consistent with that obtained by gel electrophoresis.     

Molecular requirements for degradation of a modified sense RNA strand by Escherichia coli ribonuclease H1

The structural requirements for DNA/RNA hybrids to be suitable substrates for RNase H1 are well described; however the tolerance level of this enzyme towards modifications that do not alter the duplex conformation is not clearly understood, especially with respect to the sense RNA strand. In order to investigate the molecular requirements of Escherichia coli RNase H1 (termed RNase H1 here) with respect to the sense RNA strand, we synthesized a series of oligonucleotides containing 2'-deoxy-2'-fluoro-beta-D-ribose (2'F-RNA) as a substitute for the natural beta-D-ribose sugars found in RNA. Our results from a series of RNase H1 binding and cleavage studies indicated that 2'F-RNA/DNA hybrids are not substrates of RNase H1 and ultimately led to the conclusion that the 2'-hydroxyl moiety of the RNA strand in a DNA/RNA hybrid is required for both binding and hydrolysis by RNase H1. Through the synthesis of a series of chimeric sense oligonucleotides of mixed RNA and 2'F-RNA composition, the gap requirements of RNase H1 within the sense strand were examined. Results from these studies showed that RNase H1 requires at least five or six natural RNA residues within the sense RNA strand of a hybrid substrate for both binding and hydrolysis. The RNase H1-mediated degradation patterns of these hybrids agree with previous suggestions on the processivity of RNase H1, mainly that the binding site is located 5' to the catalytic site with respect to the sense strand. They also suggest, however, that the binding and catalytic domains of RNase H1 might be closer than has been previously suggested. In addition to the above, physicochemical studies have revealed the thermal stabilities and relative conformations of these modified heteroduplexes under physiological conditions. These findings offer further insights into the physical binding and catalytic properties of the RNase H1-substrate interaction, and have been incorporated into a general model summarizing the mechanism of action of this unique enzyme.     

Identification of a subunit assembly domain in the alpha subunit of Escherichia coli RNA polymerase

The alpha subunit of Escherichia coli DNA-dependent RNA polymerase is encoded by the rpoA gene and plays a major role in enzyme assembly. A set of C-terminal deletion mutations of the rpoA gene was constructed. The results of mixed reconstitution experiments in vitro, using the truncated alpha polypeptides encoded by the rpoA deletion mutants, suggest that the amino-terminal two-thirds of alpha subunit is sufficient for the formation of pseudo-core complexes containing both beta and beta' subunits.     

Impairment of ribosomal subunit synthesis in aminoglycoside-treated ribonuclease mutants of Escherichia coli

The bacterial ribosome is an important target for many antimicrobial agents. Aminoglycoside antibiotics bind to both 30S and 50S ribosomal subunits, inhibiting translation and subunit formation. During ribosomal subunit biogenesis, ribonucleases (RNases) play an important role in rRNA processing. E. coli cells deficient for specific processing RNases are predicted to have an increased sensitivity to neomycin and paromomycin. Four RNase mutant strains showed an increased growth sensitivity to both aminoglycoside antibiotics. E. coli strains deficient for the rRNA processing enzymes RNase III, RNase E, RNase G or RNase PH showed significantly reduced subunit amounts after antibiotic treatment. A substantial increase in a 16S RNA precursor molecule was observed as well. Ribosomal RNA turnover was stimulated, and an enhancement of 16S and 23S rRNA fragmentation was detected in E. coli cells deficient for these enzymes. This work indicates that bacterial RNases may be novel antimicrobial targets.     

Analysis of the gene encoding the RNA subunit of ribonuclease P from cyanobacteria.

The genes encoding the RNA subunit of ribonuclease P from the unicellular cyanobacterium Synechocystis sp. PCC 6803, and from the heterocyst-forming strains Anabaena sp. PCC 7120 and Calothrix sp. PCC 7601 were cloned using the homologous gene from Anacystis nidulans (Synechococcus sp. PCC 6301) as a probe. The genes and the flanking regions were sequenced. The genes from Anabaena and Calothrix are flanked at their 3'-ends by short tandemly repeated repetitive (STRR) sequences. In addition, two other sets of STRR sequences were detected within the transcribed regions of the Anabaena and Calothrix genes, increasing the length of a variable secondary structure element present in many RNA subunits of ribonuclease P from eubacteria. The ends of the mature RNAs were determined by primer extension and RNase protection. The predicted secondary structure of the three RNAs studied is similar to that of Anacystis and although some idiosyncrasies are observed, fits well with the eubacterial consensus.     

The bacteriophage P1 hot gene product can substitute for the Escherichia coli DNA polymerase III {theta} subunit

The theta subunit (holE gene product) of Escherichia coli DNA polymerase (Pol) III holoenzyme is a tightly bound component of the polymerase core. Within the core (alpha-epsilon-theta), the alpha and epsilon subunits carry the DNA polymerase and 3' proofreading functions, respectively, while the precise function of theta is unclear. holE homologs are present in genomes of other enterobacteriae, suggestive of a conserved function. Putative homologs have also been found in the genomes of bacteriophage P1 and of certain conjugative plasmids. The presence of these homologs is of interest, because these genomes are fully dependent on the host replication machinery and contribute few, if any, replication factors themselves. To study the role of these theta homologs, we have constructed an E. coli strain in which holE is replaced by the P1 homolog, hot. We show that hot is capable of substituting for holE when it is assayed for its antimutagenic action on the proofreading-impaired dnaQ49 mutator, which carries a temperature-sensitive epsilon subunit. The ability of hot to substitute for holE was also observed with other, although not all, dnaQ mutator alleles tested. The data suggest that the P1 hot gene product can substitute for the theta subunit and is likely incorporated in the Pol III complex. We also show that overexpression of either theta or Hot further suppresses the dnaQ49 mutator phenotype. This suggests that the complexing of dnaQ49-epsilon with theta is rate limiting for its ability to proofread DNA replication errors. The possible role of hot for bacteriophage P1 is discussed.     

Studies of the RNA degradosome-organizing domain of the Escherichia coli ribonuclease RNase E

The hydrolytic endoribonuclease RNase E, which is widely distributed in bacteria and plants, plays key roles in mRNA degradation and RNA processing in Escherichia coli. The enzymatic activity of RNase E is contained within the conserved amino-terminal half of the 118 kDa protein, and the carboxy-terminal half organizes the RNA degradosome, a multi-enzyme complex that degrades mRNA co-operatively and processes ribosomal and other RNA. The study described herein demonstrates that the carboxy-terminal domain of RNase E has little structure under native conditions and is unlikely to be extensively folded within the degradosome. However, three isolated segments of 10-40 residues, and a larger fourth segment of 80 residues, are predicted to be regions of increased structural propensity. The larger of these segments appears to be a protein-RNA interaction site while the other segments possibly correspond to sites of self-recognition and interaction with the other degradosome proteins. The carboxy-terminal domain of RNase E may thus act as a flexible tether of the degradosome components. The implications of these and other observations for the organization of the RNA degradosome are discussed.     

Product release is a rate-limiting step during cleavage by the catalytic RNA subunit of Escherichia coli RNase P.

The kinetic constants for cleavage of the tRNA(Tyr)Su3 precursor by the M1 RNA of E. coli RNase P were determined in the absence and presence of the C5 protein under single and multiple (steady state) turnover conditions. The rate constant of cleavage in the reaction catalyzed by M1 RNA alone was 5 times higher in single turnover than in multiple turnovers, suggesting that a rate-limiting step is product release. Cleavage by M1 RNA alone and by the holoenzyme under identical buffer conditions demonstrated that C5 facilitated product release. Addition of different product-like molecules under single turnover reaction conditions inhibited cleavage both in the absence and presence of C5. In the presence of C5, the Ki value for matured tRNA was approximately 20 times higher than in its absence, suggesting that C5 also reduces the interaction between the 5'-matured tRNA and the enzyme. In a growing cell the number of tRNA molecules is approximately 1000 times higher than the number of RNase P molecules. A 100-fold excess of matured tRNA over enzyme clearly inhibited cleavage in vitro. We discuss the possibility that RNase P is involved in the regulation of tRNA expression under certain growth conditions.     

Multiple binding modes of substrate to the catalytic RNA subunit of RNase P from Escherichia coli.

M1 RNA that contained 4'-thiouridine was photochemically cross-linked to different substrates and to a product of the reaction it governs. The locations of the cross-links in these photochemically induced complexes were identified. The cross-links indicated that different substrates share some contacts but have distinct binding modes to M1 RNA. The binding of some substrates also results in a substrate-dependent conformational change in the enzymatic RNA, as evidenced by the appearance of an M1 RNA intramolecular cross-link. The identification of the cross-links between M1 RNA and product indicate that they are shared with only one of the three cross-linked E-S complexes that were identified, an indication of noncompetitive inhibition by the product. We also examined whether the cross-linked complexes between M1 RNA and substrate(s) or product are altered in the presence of the enzyme's protein cofactor (C5 protein) and in the presence of different concentrations of divalent metal ions. C5 protein enhanced the yield of certain M1 RNA-substrate cross-linked complexes for both wild-type M1 RNA and a deletion mutant of M1 RNA (delta[273-281]), but not for the M1 RNA-product complex. High concentrations of Mg2+ increased the yield of all M1 RNA-substrate complexes but not the M1 RNA-product complex.