Sok antisense RNA from plasmid R1 is functionally inactivated by RNase E and polyadenylated by poly(A) polymerase I

The hok/sok system of plasmid R1, which mediates plasmid stabilization by the killing of plasmid-free cells, codes for two RNA species, Sok antisense RNA and hok mRNA. Sok RNA, which is unstable, inhibits translation of the stable hok mRNA. The 64 nt Sok RNA folds into a single stem-loop domain with an 11 nt unstructured 5' domain. The initial recognition reaction between Sok RNA and hok mRNA takes place between the 5' domain and the complementary region in hok mRNA. In this communication we examine the metabolism of Sok antisense RNA. We find that RNase E cleaves the RNA 6 nt from its 5' end and that this cleavage initiates Sok RNA decay. The RNase E cleavage occurs in the part of Sok RNA that is responsible for the initial recognition of the target loop in hok mRNA and thus leads to functional inactivation of the antisense. The major RNase E cleavage product (denoted pSok_-6) is rapidly degraded by polynucleotide phosphorylase (PNPase). Thus, the RNase E cleavage tags pSok₋₆ for further rapid degradation by PNPase from its 3' end. We also show that Sok RNA is polyadenylated by poly(A) polymerase I (PAP I), and that the poly(A)-tailing is prerequisite for the rapid 3'-exonucleolytic degradation by PNPase.     

Interplay among processing and degradative enzymes and a precursor ribonucleic acid in the selective maturation and maintenance of ribonucleic acid molecules

In order to understand why the first tRNA (tRNAGln) in the T4 tRNA gene cluster is not produced when T4 infects an RNase III- mutant of Escherichia coli, RNA metabolism was analyzed in RNase III- RNase P- (rnc, rnp) cells infected with bacteriophage T4. After such an infection a new dimeric precursor RNA molecule of tRNAGln and tRNALeu has been identified and analyzed. This molecule is structurally very similar to K band RNA that accumulates in rnc+ rnp strains. It is four nucleotides shorter than K RNA at the 5' end. This molecule like K RNA contains two RNase P processing sites at the 5' ends of each tRNA. Both sites are accessible to RNase P. However, while in the K RNA the site at the 5' end of tRNALeu (the site in the middle of the substrate) is more efficiently cleaved than the other site, this differential is even increased in the Ks (K like) molecule. This difference is sufficiently large that in vivo in the RNase III- strain the smaller precursor of tRNAGln is degraded rather than being matured to tRNAGln by RNase P. This information contributes to the elucidation of the key role of RNase III in the processing of T4 tRNA. It shows the dependence of RNase P activity at the 5' end of tRNAGln on a correct and specific cleavage by RNase III at a position six nucleotides proximal to the RNase P site, and it explains why in the absence of RNase III the first tRNA in the T4 tRNA cluster, tRNAGln, does not accumulate.     

Erythromycin-induced ribosome stalling and RNase J1-mediated mRNA processing in Bacillus subtilis

Addition of erythromycin (Em) to a Bacillus subtilis strain carrying the ermC gene results in ribosome stalling in the ermC leader peptide coding sequence. Using Δ ermC , a deletion derivative of ermC that specifies the 254 nucleotide Δ ermC mRNA, we showed previously that ribosome stalling is concomitant with processing of Δ ermC mRNA, generating a 209 nucleotide RNA whose 5' end maps to codon 5 of the Δ ermC coding sequence. Here we probed for peptidyl-tRNA to show that ribosome stalling occurs after incorporation of the amino acid specified by codon 9. Thus, cleavage upstream of codon 5 is not an example of 'A-site cleavage' that has been reported for Escherichia coli . Analysis of Δ ermC mRNA processing in endoribonuclease mutant strains showed that this processing is RNase J1-dependent. Δ ermC mRNA processing was inhibited by the presence of stable secondary structure at the 5' end, demonstrating 5'-end dependence, and was shown to be a result of RNase J1 endonuclease activity, rather than 5'-to-3' exonuclease activity. Examination of processing in derivatives of Δ ermC that had codons inserted upstream of the ribosome stalling site revealed that Em-induced ribosome stalling can occur considerably further from the start codon than would be expected based on previous studies.     

Degradation of Escherichia coli uncB mRNA by multiple endonucleolytic cleavages.

The mechanism of segmental decay of the uncB sequence near the 5' end of the 7-kb Escherichia coli unc operon mRNA was investigated. Northern (RNA) blots of mRNA expressed from a plasmid carrying the uncBE portion of the operon revealed that the uncB message was rapidly degraded by multiple internal cleavages which resulted in the formation of at least five discrete species having a common 3' end. Turnover studies indicated that processing rapidly converted all species to the smallest. Identification of the 5' ends by primer extension analysis revealed that the cleavages were made either in the uncB coding region or in the intercistronic region between uncB and uncE, the latter being the most 3' cleavage. An rne mutant strain contained much higher levels of the uncBE message, implying that RNase E, the product of the rne gene, is essential for the normal degradation of uncB, and a number of the 5' ends were not detected in the rne mutant. The cleavage sites in chromosomally encoded unc mRNA were also identified by primer extension. These studies reveal that the segmental decay of the uncB region of unc mRNA occurs rapidly through a series of endonucleolytic cleavages. The rapid decay of uncB is expected to play a role in limiting expression of this gene relative to that of the other genes of the operon.     

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.     

A novel function of RNase P from Escherichia coli: processing of a suppressor tRNA precursor.

The leuX gene of Escherichia coli codes for a suppressor tRNA and forms a single gene operon containing its own promoter and Q-independent terminator. An analysis of the in vitro processing of leuX precursor revealed that the processing of the 5' end took place in a single-step reaction catalysed by RNase P while the 3' processing involved two successive reactions. The endonucleolytic cleavage activity of the 3' precursor sequence was found to copurify with RNase P. Heat inactivation of thermosensitive RNase P from two independent E. coli mutants abolished the cleavage activity of both the 5' and 3' ends. These results altogether suggest that RNase P carries the activity of 3' end cleavage as well as that of 5' processing. In the presence of Mg2+ alone, the leuX precursor was found to be self-cleaved at a site approximately 13 nt inside from the 5' end of mature tRNA. The self-cleaved precursor tRNA was no longer processed by the 3' endonuclease, suggesting that the 3' endonuclease recognizes a specific conformation of the precursor tRNA for action.     

A site in a tRNA precursor that can be processed by the whole RNase P enzyme but not by the RNA alone

A precursor molecule for 10 Sb RNA, the RNA moiety of the RNA processing enzyme RNase P, was purified, characterized for enzymatic activity, and compared to 10 Sb RNA and to RNase P. In these studies the K RNA, a dimeric precursor of tRNAGln-tRNALeu, coded by bacteriophage T4, was used as a substrate. This precursor contains two RNase P cleavage sites, one at each 5' end of the two tRNAs. The precursor 10 Sb and 10 Sb RNAs have the capacity to cleave the precursor tRNA molecule but only at the 5' end of tRNALeu, not at the 5' end of tRNAGln. Even when a substrate was prepared that contained only one site for RNase P (the one next to tRNAGln), this substrate was not cleaved by the RNA alone while the whole enzyme was effective in processing this substrate. The possible function of the protein of RNase P in the enzymatic reaction is discussed.     

Regions of RNase E important for 5'-end-dependent RNA cleavage and autoregulated synthesis

RNase E is an important regulatory enzyme that plays a key role in RNA processing and degradation in Escherichia coli. Internal cleavage by this endonuclease is accelerated by the presence of a monophosphate at the RNA 5' end. Here we show that the preference of E. coli RNase E for 5'-monophosphorylated substrates is an intrinsic property of the catalytically active amino-terminal half of the enzyme and does not require the carboxy-terminal region. This property is shared by the related E. coli ribonuclease CafA (RNase G) and by a cyanobacterial RNase E homolog derived from Synechocystis, indicating that the 5'-end dependence of RNase E is a general characteristic of members of this ribonuclease family, including those from evolutionarily distant species. Although it is dispensable for 5'-end-dependent RNA cleavage, the carboxy-terminal half of RNase E significantly enhances the ability of this ribonuclease to autoregulate its synthesis in E. coli. Despite similarities in amino acid sequence and substrate specificity, CafA is unable to replace RNase E in sustaining E. coli cell growth or in regulating RNase E production, even when overproduced sixfold relative to wild-type RNase E levels.     

Autoregulation of RNase III operon by mRNA processing.

RNase III has been implicated in the control of gene expression by the processing of mRNA. We have found that the rnc operon is autoregulated; rnc- mutant strains oversynthesize the operon's mRNA and protein products. A site in the 5'-noncoding region of the operon's message is cleaved by RNase III. This site-specific cleavage appears to be the initial step in the functional inactivation of the message, since the half-life of the cut message is dramatically shorter than that of the uncut message.