DNA sequencing and expression of the gene rnb encoding Escherichia coli ribonuclease II

The Escherichia coli ribonuclease II (RNase II) is an exonuclease involved in mRNA degradation that hydrolyses single-stranded polyribonucleotides processively in the 3′ to 5′ direction. Sequencing of a 2.2 kb Msel–Rsal fragment containing the rnb gene revealed an open reading frame of 1794 nucleotides that encodes a protein of 598 amino acid residues, whose calculated molecular mass is 67 583 Da. This value is in good agreement with that obtained by sodium dodecyl sulphate/ polyacrylamide gel electrophoresis of polypeptides synthesized by expression with the T7 RNA polymerase/promoter system. This system was also used to confirm the correct orientation of rnb. Translation initiation was confirmed by rnb–lacZ fusions. The mRNA start site was determined by S1 nuclease mapping. Two E. coli mutants harbouring different rnb alleles deficient in RNase II activity were complemented with the expressed fragment carrying the rnb gene.     

Precise physical mapping of theEscherichia coli rnb gene, encoding ribonuclease II

Ribonuclease II (encoded byrnb) is one of the two main exonucleases involved in mRNA degradation inEscherichia coli. We report the precise physical mapping ofrnb to 29 min on the chromosomal map in the vicinity ofpyrF, and clarify the genetic and physical maps of thisE. coli chromosomal region. The results were confirmed by the construction of a strain partially deleted forrnb.     

A protein complex mediating mRNA degradation in Escherichia coli

mRNA degradation in Escherichia coli is mediated by a combination of exo- and endoribolucleases. We present evidence for a multiprotein complex which includes at least two enzymes that play important roles in mRNA degradation: the exoribonuclease poly-nucleotide phosphorylase (PNPase) and the endorlbo-nuclease RNase E. An activity which impedes the processive activity of PNPase at stem-loop structures also appears to be associated with the complex. This complex is estimated to have a molecular mass of about 500 kDa and includes several additional poly-peptides whose functions are unknown. The identification of a complex which includes several activities associated with mRNA degradation has implications for the mechanisms and co-ordinated control of mRNA degradation.     

Replication initiator protein mRNA of ColE2 plasmid and its antisense regulator RNA are under the control of different degradation pathways

Replication of the ColE2 plasmid requires a plasmid-coded initiator protein (Rep). Rep expression is controlled by antisense RNA (RNAI) against the Rep mRNA at a translational step. In this paper, we examined the effects of host RNA degradation enzymes on the degradation process of the Rep mRNA and its degradation intermediates especially those carrying the 5' untranslated region. We showed that the Rep mRNA is subjected to complex degradation pathways involving at least RNase I, RNase II, RNase III, RNase E, RNase G and PNPase. RNase II acts as a major exoribonuclease and PNPase plays a minor role. We also showed that the PcnB (polyA polymerase I) plays only a minor role in the Rep mRNA degradation process. The RNA degradation pathways of the Rep mRNA and RNAI of the ColE2 plasmid are quite different. Based on these results, we speculate that the ColE2 Rep mRNA and RNAI are endowed with individual RNA half lives required for the efficient copy number control by being subjected to different RNA degradation systems.     

Novel role for RNase PH in the degradation of structured RNA

Escherichia coli contains multiple 3' to 5' RNases, of which two, RNase PH and polynucleotide phosphorylase (PNPase), use inorganic phosphate as a nucleophile to catalyze RNA cleavage. It is known that an absence of these two enzymes causes growth defects, but the basis for these defects has remained undefined. To further an understanding of the function of these enzymes, the degradation pattern of different cellular RNAs was analyzed. It was observed that an absence of both enzymes results in the appearance of novel mRNA degradation fragments. Such fragments were also observed in strains containing mutations in RNase R and PNPase, enzymes whose collective absence is known to cause an accumulation of structured RNA fragments. Additional experiments indicated that the growth defects of strains containing RNase R and PNPase mutations were exacerbated upon RNase PH removal. Taken together, these observations suggested that RNase PH could play a role in structured RNA degradation. Biochemical experiments with RNase PH demonstrated that this enzyme digests through RNA duplexes of moderate stability. In addition, mapping and sequence analysis of an mRNA degradation fragment that accumulates in the absence of the phosphorolytic enzymes revealed the presence of an extended stem-loop motif at the 3' end. Overall, these results indicate that RNase PH plays a novel role in the degradation of structured RNAs and provides a potential explanation for the growth defects caused by an absence of the phosphorolytic RNases.     

Decay of the IS 10 antisense RNA by 3' exoribonucleases: evidence that RNase II stabilizes RNA-OUT against PNPase attack

RNA-OUT, the 69-nucleotide antisense RNA that regulates Tn 10/IS 10 transposition folds into a simple stem-loop structure. The unusually high metabolic stability of RNA-OUT is dependent, in part, on the integrity of its stem-domain: mutations that disrupt stem-domain structure (Class II mutations) render RNA-OUT unstable, and restoration of structure restores stability. Indeed, there is a strong correlation between the thermodynamic and metabolic stabilities of RNA-OUT. We show here that stem-domain integrity determines RNA-OUT's resistance to 3’exoribonucleolytic attack: Class II mutations are almost completely suppressed in Escherichia coli cells lacking its principal 3′ exoribonucleases, ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase). RNase II and PNPase are individually able to degrade various RNA-OUT species, albeit with different efficiencies: RNA-OUT secondary structure provides greater resistance to RNase II than to PNPase. Surprisingly, RNA-OUT is threefold more stable in wild-type cells than in cells deficient for RNase II activity, suggesting that RNase II somehow lessens RNPase attack on RNA-OUT. We discuss how this might occur. We also show that wild-type RNA-OUT stability changes only twofold across the normal range of physiological growth temperatures (30–44°C) in wild-type cells, which has important implications for IS 10 biology.     

Precise physical mapping of theEscherichia coli rnb gene,encoding ribonuclease II

Ribonuclease II (encoded byrnb) is one of the two main exonucleases involved in mRNA degradation inEscherichia coli. We report the precise physical mapping ofrnb to 29 min on the chromosomal map in the vicinity ofpyrF, and clarify the genetic and physical maps of thisE. coli chromosomal region. The results were confirmed by the construction of a strain partially deleted forrnb.     

An important role for RNase R in mRNA decay

mRNA decay is a major determinant of gene expression. In Escherichia coli, message degradation initiates with an endoribonucleolytic cleavage followed by exoribonuclease digestion to generate 5'-mononucleotides. Although the 3' to 5' processive exoribonucleases, PNPase and RNase II, have long been considered to be mediators of this digestion, we show here that another enzyme, RNase R, also participates in the process. RNase R is particularly important for removing mRNA fragments with extensive secondary structure, such as those derived from the many mRNAs that contain REP elements. In the absence of RNase R and PNPase, REP-containing fragments accumulate to high levels. RNase R is unusual among exoribonucleases in that, by itself, it can digest through extensive secondary structure provided that a single-stranded binding region, such as a poly(A) tail, is present. These data demonstrate that RNase R, which is widespread in prokaryotes and eukaryotes, is an important participant in mRNA decay.     

The effects of RNA degradation enzymes on antisense RNAI controlling ColE2 plasmid copy number

Replication of the ColE2 plasmid requires a plasmid-coded initiator protein (Rep). Rep expression is controlled by antisense RNA (RNAI), which prevents the Rep mRNA translation. In this paper, we examined the effects of RNA degradation enzymes on the degradation pathways of RNAI of the ColE2 plasmid. In the DeltapcnB strain lacking the poly(A) polymerase I (PAP I) the RNAI degradation intermediate (RNAI(*)) accumulates much more than that in the wt strain. RNAI(*) is produced by the RNase E cleavage. RNase II and PNPase are involved in further degradation of RNAI(*) and PAP I is necessary for efficient degradation. The degradation process of ColE2 RNAI is similar to those of R1 CopA RNA and ColE1 RNAI, although the nucleotide sequences and fine secondary structures of these three RNAs are different. ColE2 RNAI is cleaved at multiple positions in the 5' end region by RNase E. The degradation pathway of ColE2 RNAI shown here is quite different from that of the ColE2 Rep mRNA which we have previously reported. In the DeltapcnB strain used for RNA analysis the copy number of the ColE2 plasmid decreases to about a half as compared with that in the isogenic wt strain.     

RNase activity of polynucleotide phosphorylase is critical at low temperature in Escherichia coli and is complemented by RNase II

In Escherichia coli, the cold shock response is exerted upon a temperature change from 37°C to 15°C and is characterized by induction of several cold shock proteins, including polynucleotide phosphorylase (PNPase), during acclimation phase. In E. coli, PNPase is essential for growth at low temperatures; however, its exact role in this essential function has not been fully elucidated. PNPase is a 3′-to-5′ exoribonuclease and promotes the processive degradation of RNA. Our screening of an E. coli genomic library for an in vivo counterpart of PNPase that can compensate for its absence at low temperature revealed only one protein, another 3′-to-5′ exonuclease, RNase II. Here we show that the RNase PH domains 1 and 2 of PNPase are important for its cold shock function, suggesting that the RNase activity of PNPase is critical for its essential function at low temperature. We also show that its polymerization activity is dispensable in its cold shock function. Interestingly, the third 3′-to-5′ processing exoribonuclease, RNase R of E. coli, which is cold inducible, cannot complement the cold shock function of PNPase. We further show that this difference is due to the different targets of these enzymes and stabilization of some of the PNPase-sensitive mRNAs, like fis, in the Δpnp cells has consequences, such as accumulation of ribosomal subunits in the Δpnp cells, which may play a role in the cold sensitivity of this strain.     

Escherichia coli strains with modified RNase II activity: Isolation and properties

Summary In a search for Escherichia coli strains defective in ribonuclease activity, a uracil requiring strain lacking RNase I activity was fed yeast RNA as the sole source of uracil and mutants that failed to utilize yeast RNA as the sole source of uracil were isolated. Among thirty colonies thus isolated and tested three were found to contain a heat labile RNase activity in cell free extracts. (Assays were performed under conditions for RNase II.) Since no strain completely lacking this RNase activity was found, and since these three strains have reduced growth rates that seem to be caused by the same mutation that altered the RNase activity, we concluded that RNase II activity is indispensible during cell growth.     

The Escherichia coli major exoribonuclease RNase II is a component of the RNA degradosome

Multiprotein complexes that carry out RNA degradation and processing functions are found in cells from all domains of life. In Escherichia coli, the RNA degradosome, a four-protein complex, is required for normal RNA degradation and processing. In addition to the degradosome complex, the cell contains other ribonucleases that also play important roles in RNA processing and/or degradation. Whether the other ribonucleases are associated with the degradosome or function independently is not known. In the present work, IP (immunoprecipitation) studies from cell extracts showed that the major hydrolytic exoribonuclease RNase II is associated with the known degradosome components RNaseE (endoribonuclease E), RhlB (RNA helicase B), PNPase (polynucleotide phosphorylase) and Eno (enolase). Further evidence for the RNase II-degradosome association came from the binding of RNase II to purified RNaseE in far western affinity blot experiments. Formation of the RNase II–degradosome complex required the degradosomal proteins RhlB and PNPase as well as a C-terminal domain of RNaseE that contains binding sites for the other degradosomal proteins. This shows that the RNase II is a component of the RNA degradosome complex, a previously unrecognized association that is likely to play a role in coupling and coordinating the multiple elements of the RNA degradation pathways.