RNase III cleavage of Escherichia coli beta-galactosidase and tryptophan operon mRNA.

Purified RNase III of Escherichia coli cleaved the initial 479-nucleotide sequence of lac operon mRNA at four specific sites and also gave limited cleavage of trp operon mRNA. This action explains the inactivation of mRNA coding capacity by RNase III in vitro.     

Sequence dependence of substrate recognition and cleavage by yeast RNase III

Yeast Rnt1p is a member of the double-stranded RNA (dsRNA) specific RNase III family of endoribonucleases involved in RNA processing and RNA interference (RNAi). Unlike other RNase III enzymes, which recognize a variety of RNA duplexes, Rnt1p cleaves specifically RNA stems capped with the conserved AGNN tetraloop. This unusual substrate specificity challenges the established dogma for substrate selection by RNase III and questions the dsRNA contribution to recognition by Rnt1p. Here we show that the dsRNA sequence adjacent to the tetraloop regulates Rnt1p cleavage by interfering with RNA binding. In context, sequences surrounding the cleavage site directly influence the cleavage efficiency. Introduction of sequences that stabilize the RNA helix enhanced binding while reducing the turnover rate indicating that, unlike the tetraloop, Rnt1p binding to the dsRNA helix may become rate-limiting. These results suggest that Rnt1p activity is strictly regulated by a combination of primary and tertiary structural elements allowing a substrate-specific binding and cleavage efficiency.     

Analysis of mRNA decay and rRNA processing in Escherichia coli multiple mutants carrying a deletion in RNase III.

RNase III is an endonuclease involved in processing both rRNA and certain mRNAs. To help determine whether RNase III (rnc) is required for general mRNA turnover in Escherichia coli, we have created a deletion-insertion mutation (delta rnc-38) in the structural gene. In addition, a series of multiple mutant strains containing deficiencies in RNase II (rnb-500), polynucleotide phosphorylase (pnp-7 or pnp-200), RNase E (rne-1 or rne-3071), and RNase III (delta rnc-38) were constructed. The delta rnc-38 single mutant was viable and led to the accumulation of 30S rRNA precursors, as has been previously observed with the rnc-105 allele (P. Gegenheimer, N. Watson, and D. Apirion, J. Biol. Chem. 252:3064-3073, 1977). In the multiple mutant strains, the presence of the delta rnc-38 allele resulted in the more rapid decay of pulse-labeled RNA but did not suppress conditional lethality, suggesting that the lethality associated with altered mRNA turnover may be due to the stabilization of specific mRNAs. In addition, these results indicate that RNase III is probably not required for general mRNA decay. Of particular interest was the observation that the delta rnc-38 rne-1 double mutant did not accumulate 30S rRNA precursors at 30 degrees C, while the delta rnc-38 rne-3071 double mutant did. Possible explanations of these results are discussed.     

Human let-7 stem-loop precursors harbor features of RNase III cleavage products

The bidentate RNase III Dicer cleaves microRNA precursors to generate the 21–23 nt long mature RNAs. These precursors are 60–80 nt long, they fold into a characteristic stem–loop structure and they are generated by an unknown mechanism. To gain insights into the biogenesis of microRNAs, we have characterized the precise 5′ and 3′ ends of the let-7 precursors in human cells. We show that they harbor a 5′-phosphate and a 3′-OH and that, remarkably, they contain a 1–4 nt 3′ overhang. These features are characteristic of RNase III cleavage products. Since these precursors are present in both the nucleus and the cytoplasm of human cells, our results suggest that they are generated in the nucleus by the nuclear RNase III. Additionally, these precursors fit the minihelix export motif and are thus likely exported by this pathway.     

The cleavage specificity of RNase III.

We determined sites in lambda cII mRNA that are cleaved by RNase III in the presence of lambda OOP antisense RNA, using a series of OOP RNAs with different internal deletions. In OOP RNA-cII mRNA structures containing a potential region of continuous double-stranded RNA bounded by a non-complementary unpaired region, RNase III cleaved the cII mRNA at one or more preferred sites located 10 to 14 bases from the 3'-end of the region of continuous complementarity. Cleavage patterns were almost identical when the presumptive structure was the same continuously double-stranded region followed by a single-stranded bulge and a second short region of base pairing. The sequences of the new cleavage sites show generally good agreement with a consensus sequence derived from thirty-five previously determined cleavage sequences. In contrast, four 'non-sites' at which cleavage is never observed show poor agreement with this consensus sequence. We conclude that RNase III specificity is determined both by the distance from the end of continuous pairing and by nucleotide sequence features within the region of pairing.     

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.     

Escherichia coli endoribonuclease RNase E: autoregulation of expression and site-specific cleavage of mRNA

Mutations in the Escherichia coli rne (ams) gene have a general effect on the rate of mRNA decay in vivo. Using antibodies we have shown that the product of the rne gene is a polypeptide of relative mobility 180kDa. However, proteolytic fragments as small as 70kDa, which can arise during purification, also exhibit RNase E activity, in vitro studies demonstrate that the rne gene product, RNase E, is an endoribonuclease that cleaves mRNA at specific sites. RNase E cleaves rne mRNA and autoregulates the expression of the rne gene. In addition we demonstrate RNase E-dependent endonucleolytic cleavage of ompA mRNA, at a site known to be rate-determining for degradation and reported to be cieaved by RNase K. Our data are consistent with RNase K being a proteolytic fragment of RNase E.     

Functional interaction between RNase III and the Escherichia coli ribosome

Background  

RNase III is a dsRNA specific endoribonuclease which is involved in the primary processing of rRNA and several mRNA species in bacteria. Both primary structural elements and the secondary structure of the substrate RNA play a role in cleavage specificity.     

Functional Conservation of RNase III-like Enzymes: Studies on a Vibrio vulnificus Ortholog of Escherichia coli RNase III

Bacterial ribonuclease III (RNase III) belongs to the RNase III enzyme family, which plays a pivotal role in controlling mRNA stability and RNA processing in both prokaryotes and eukaryotes. In the Vibrio vulnificus genome, one open reading frame encodes a protein homologous to E. coli RNase III, designated Vv-RNase III, which has 77.9 % amino acid identity to E. coli RNase III. Here, we report that Vv-RNase III has the same cleavage specificity as E. coli RNase III in vivo and in vitro. Expressing Vv-RNase III in E. coli cells deleted for the RNase III gene (rnc) restored normal rRNA processing and, consequently, growth rates of these cells comparable to wild-type cells. In vitro cleavage assays further showed that Vv-RNase III has the same cleavage activity and specificity as E. coli RNase III on RNase III-targeted sequences of corA and mltD mRNA. Our findings suggest that RNase III-like proteins have conserved cleavage specificity across bacterial species.     

Several regions of a tRNA precursor determine the Escherichia coli RNase P cleavage site.

The RNase P cleavage reaction was studied as a function of the number of base-pairs in the acceptor-stem and/or T-stem of a natural tRNA precursor, the tRNA(Tyr)Su3 precursor. Our data suggest that the location of the Escherichia coli RNase P cleavage site does not depend merely on the lengths of the acceptor-stem and T-stem as previously suggested. Surprisingly, we find that precursors with only four base-pairs in the acceptor-stem are cleaved by M1 RNA and by holoenzyme. Furthermore, we show that both disruption of base-pairing, and alteration of the nucleotide sequence (without disruption of base-pairing) proximal to the cleavage site result in aberrant cleavage. Thus, the identity of the nucleotides near the cleavage site is important for recognition of the cleavage site rather than base-pairing. The important nucleotides are those at positions -2, -1, +1, +72, +73 and +74. We propose that the nucleotide at position +1 functions as a guiding nucleotide. These results raise the possibility that Mg2+ binding near the cleavage site is dependent on the identity of the nucleotides at these positions. In addition, we show that disruption of base-pairing in the acceptor-stem affects both Michaelis-Menten constants, Km and kcat.     

Characterization of Escherichia coli RNase PH.

We have previously shown that the orfE gene of Escherichia coli encodes RNase PH. Here we show that the OrfE protein (purified as described in the accompanying paper) (Jensen, K. F., Andersen, J. T., and Poulsen, P. (1992) J. Biol. Chem. 267, 17147-17152) has both the degradative and synthetic activities of RNase PH. This highly purified protein was used to characterize the enzymatic and structural properties of RNase PH. The enzyme requires a divalent cation and phosphate for activity, the latter property indicating that RNase PH is exclusively a phosphorolytic enzyme. Among tRNA-type substrates, the enzyme is most active against synthetic tRNA precursors containing extra residues following the -CCA sequence, and it can act on these molecules to generate mature tRNA with amino acid acceptor activity; 3'-phosphoryl-terminated molecules are not active as substrates. The equilibrium constant for RNase PH is near unity, suggesting that at the phosphate concentration present in vivo, the enzyme would participate in RNA degradation. The synthetic reaction of RNase PH displays a nonlinear response to increasing enzyme concentrations, and this may be due to self-aggregation of the protein. Higher order multimers of RNase PH could be detected by gel filtration at higher protein concentrations and by protein cross-linking. The possible role of RNase PH in tRNA processing is discussed.     

RNase III controls the degradation of corA mRNA in Escherichia coli

In Escherichia coli, the corA gene encodes a transporter that mediates the influx of Co(2+), Mg(2+), and Ni(2+) into the cell. During the course of experiments aimed at identifying RNase III-dependent genes in E. coli, we observed that steady-state levels of corA mRNA as well as the degree of cobalt influx into the cell were dependent on cellular concentrations of RNase III. In addition, changes in corA expression levels by different cellular concentrations of RNase III were closely correlated with degrees of resistance of E. coli cells to Co(2+) and Ni(2+). In vitro and in vivo cleavage analyses of corA mRNA identified RNase III cleavage sites in the 5'-untranslated region of the corA mRNA. The introduction of nucleotide substitutions at the identified RNase III cleavage sites abolished RNase III cleavage activity on corA mRNA and resulted in prolonged half-lives of the mRNA, which demonstrates that RNase III cleavage constitutes a rate-determining step for corA mRNA degradation. These findings reveal an RNase III-mediated regulatory pathway that functions to modulate corA expression and, in turn, the influx of metal ions transported by CorA in E. coli.     

Different cleavage specificities of RNases III from Rhodobacter capsulatus and Escherichia coli.

23S rRNA in Rhodobacter capsulatus shows endoribonuclease III (RNase III)-dependent fragmentation in vivo at a unique extra stem-loop extending from position 1271 to 1331. RNase III is a double strand (ds)-specific endoribonuclease. This substrate preference is mediated by a double-stranded RNA binding domain (dsRBD) within the protein. Although a certain degree of double strandedness is a prerequisite, the question arises what structural features exactly make this extra stem-loop an RNase III cleavage site, distinguishing it from the plethora of stem-loops in 23S rRNA? We used RNase III purified from R.capsulatus and Escherichia coli, respectively, together with well known substrates for E.coli RNase III and RNA substrates derived from the special cleavage site in R.capsulatus 23S rRNA to study the interaction between the Rhodobacter enzyme and the fragmentation site. Although both enzymes are very similar in their amino acid sequence, they exhibit significant differences in binding and cleavage of these in vitro substrates.     

Expression of double-stranded-RNA-specific RNase III of Escherichia coli is lethal to Saccharomyces cerevisiae.

The gene for the double-stranded RNA (dsRNA)-specific RNase III of Escherichia coli was expressed in Saccharomyces cerevisiae to examine the effects of this RNase activity on the yeast. Induction of the RNase III gene was found to cause abnormal cell morphology and cell death. Whereas double-stranded killer RNA is degraded by RNase III in vitro, killer RNA, rRNA, and some mRNAs were found to be stable in vivo after induction of RNase III. Variants selected for resistance to RNase III induction were isolated at a frequency of 4 X 10(-5) to 5 X 10(-5). Ten percent of these resistant strains had concomitantly lost the capacity to produce killer toxin and M dsRNA while retaining L dsRNA. The genetic alteration leading to RNase resistance was localized within the RNase III-coding region but not in the yeast chromosome. These results indicate that S. cerevisiae contains some essential RNA which is susceptible to E. coli RNase III.     

Determinants of Escherichia coli RNase P cleavage site selection: a detailed in vitro and in vivo analysis.

The location of the Escherichia coli RNase P cleavage site was studied both in vitro and in vivo. We show that selection of the cleavage site is dependent on the nucleotide at the cleavage site and the length of the acceptor-stem. Within the acceptor-stem the number of nucleotides on the 5'-half of the acceptor-stem appears to be the important determinant, rather than the number of base pairs in the acceptor-stem. We also demonstrate that the length of the T-stem and a G to C substitution at position 57 in the tRNA(Tyr)Su3 precursor influence the location of the cleavage site under certain conditions. With respect to the function of the subunits of RNase P our data suggest that the nucleotide at position 333 in M1 RNA, and the C5 protein, are important for the identification of the cleavage site.