Polyadenylation can regulate ColE1 type plasmid copy number independently of any effect on RNAI decay by decreasing the interaction of antisense RNAI with its RNAII target

Replication of ColE1-type plasmids is regulated by RNAI, an antisense RNA that interacts with the replication pre-primer, RNAII. Exonucleolytic attack at the 3' end of RNAI is impeded in pcnB mutant bacteria, which lack poly(A) polymerase I-the principal RNA polyadenylase of E. coli; this leads to accumulation of an RNAI decay intermediate (RNAI(-5)) and dramatic reduction of the plasmid copy number. Here, we report that polyadenylation can also affect RNAI-mediated control of plasmid DNA replication by inhibiting interaction of RNAI(-5) with RNAII. We show that mutation of the host pcnB gene profoundly affects the plasmid copy number, even under experimental conditions that limit the effects of polyadenylation on RNAI(-5) decay. Moreover, poly(A) tails interfere with RNAI/RNAII interaction in vitro without producing any detectable alteration of RNAI secondary structure. Our results establish the existence of a previously undetected mechanism by which RNA polyadenylation can control plasmid copy number.     

Regulation of plasmid R1 replication: PcnB and RNase E expedite the decay of the antisense RNA, CopA

The replication frequency of plasmid R1 is controlled by an unstable antisense RNA, CopA, which, by binding to its complementary target, blocks translation of the replication rate-limiting protein RepA. Since the degree of inhibition is directly correlated with the intracellular concentration of CopA, factors affecting CopA turnover can also alter plasmid copy number. We show here that PcnB (PAP I — a poly(A)polymerase of Escherichia coli  ) is such a factor. Previous studies have shown that the copy number of ColE1 is decreased in pcnB mutant strains because the stability of the RNase E processed form of RNAI, the antisense RNA regulator of ColE1 replication, is increased. We find that, analogously, the twofold reduction in R1 copy number caused by a pcnB lesion is associated with a corresponding increase in the stability of the RNase E-generated 3' cleavage product of CopA. These results suggest that CopA decay is initiated by RNase E cleavage and that PcnB is involved in the subsequent rapid decay of the 3' CopA stem-loop segment. We also find that, as predicted, under conditions in which CopA synthesis is unaffected, pcnB mutation reduces RepA translation and increases CopA stability to the same extent.     

Real time analysis of the RNAI-RNAII-Rop complex by surface plasmon resonance: from a decaying surface to a standard kinetic analysis

RNA loop-loop complexes are motifs that regulate biological functions in both prokaryotic and eukaryotic organisms. In E. coli, RNAI, an antisense RNA encoded by the ColE1 plasmid, regulates the plasmid replication by recognizing through loop-loop interactions RNAII, the RNA primer that binds to the plasmidic DNA to initiate the replication. Rop, a plasmid-encoded homodimeric protein interacts with this transient RNAI-RNAII kissing complex. A surface plasmon resonance (SPR)-based biosensor was used to investigate this protein-nucleic acid ternary complex, at 5 degrees C, in experimental conditions such as the protein binds either to the loop-loop complex while it dissociates or to a saturated stable RNAI-RNAII surface. The results show that RNAI hairpin dissociates from the RNAII surface 110 times slower in the presence of Rop than in its absence. Rop binds to RNAI-RNAII with an on-rate of 3.6 x 10(6) M(-1) s(-1) and an off-rate of 0.11 s(-1), resulting in a binding equilibrium constant equal to 31 nM. A Scatchard-plot analysis of the interaction monitored by SPR confirms a 1:1 complex of Rop and RNAI-RNAII as observed for non-natural Rop-loop-loop complexes.     

Control of ColE1 replication: low affinity specific binding of Rop (Rom) to RNAI and RNAII.

We have studied the interactions between the three molecules Rop, RNAI and RNAII that are involved in the regulatory mechanism controlling the replication of ColE1 plasmids. We show that it is possible to purify the two RNA molecules by passing an RNA mixture through an affinity column containing Rop immobilized to a solid support. The dissociation constants of the Rop-RNAI and Rop-RNAII complexes are of the order of 10(-4) M, several orders of magnitude higher than dissociation constants of stable protein-nucleic acid complexes (10(-10) M in the lambda repressor system). Although complete RNAI molecules have higher affinity, stem-and-loop I alone can also bind Rop, suggesting that this structure plays an important role in the interaction. Rop protects the stems of RNAI and RNAII from digestion by RNases while the sensitivity of the loops to digestion by RNase T1 is not affected by high concentrations of Rop. We propose a model for Rop-RNAI/RNAII interaction in which the dimeric protein acts as an adaptor between stem structures to position the two RNAs in the correct position for loop interaction.     

The pcnB gene of Escherichia coli, which is required for ColE1 copy number maintenance, is dispensable.

The pcnB gene product of Escherchia coli is required for copy number maintenance of plasmids related to ColE1 and also for that of the IncFII plasmid R1. Because PcnB is similar to the tRNA-binding protein tRNA nucleotidyltransferase, we have suggested that the protein would be required only for processes in which an RNA is a prominent regulatory component. This appears to be so; strains deleted for pcnB, although defective in ColE1 and R1 plasmid maintenance, maintain the iteron-regulated plasmids F and P1 normally. We also find that strains deleted for pcnB grow normally, demonstrating that PcnB has no essential cellular role under the conditions tested and suggesting that regulation by antisense RNAs similar to RNAI has no critical role in any essential host process. We confirm by immunological tests that PcnB is likely to be the commercially available enzyme poly(A) polymerase.     

Suppression of RNA interference by adenovirus virus-associated RNA

We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses defective in virus-associated (VA) RNA expression. The results show that the virus-associated RNAs, VA RNAI and VA RNAII, function as suppressors of RNAi by interfering with the activity of Dicer. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. Further, we show that VA RNAI and VA RNAII are processed by Dicer, both in vitro and during a lytic infection, and that the resulting short interfering RNAs (siRNAs) are incorporated into active RISC. Dicer cleaves the terminal stem of both VA RNAI and VA RNAII. However, whereas both strands of the VA RNAI-specific siRNA are incorporated into RISC, the 3' strand of the VA RNAII-specific siRNA is selectively incorporated during a lytic infection. In summary, our work shows that adenovirus suppresses RNAi during a lytic infection and gives insight into the mechanisms of RNAi suppression by VA RNA.     

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.     

A possible role for the pcnB gene product of Escherichia coli in modulating RNA: RNA interactions.

Summary The sequence of the PcnB protein of Escherichia coli, a protein required for copy number maintenance of ColE1-related plasmids, was compared with the PIR sequence database. Strong local similarities to the sequence of the E. coli protein tRNA nucleotidyltransferase were found. Since a substrate of the latter protein, tRNA, structurally resembles the RNAs that control ColE1 copy number we believe that we may have identified a region in PcnB that interacts with these RNAs. Consistent with this idea is our observation that PcnB is required for the replication of R1, a plasmid whose replication is also regulated by a small RNA.     

The rate of processing and degradation of antisense RNAI regulates the replication of ColE1-type plasmids in vivo.

We show that the rate of degradation of RNAI, an anti-sense repressor of the replication primer RNAII, is a key element of control in the replication of ColE1-type plasmids in vivo. Cleavage of RNAI by RNAase E, a ribosomal RNA-processing enzyme encoded or controlled by the rne (also known as ams) locus, relieves repression by endonucleolytically converting RNAI to a very rapidly decaying product, pRNAI-5. A 5' triphosphate-terminated homolog of pRNAI-5 is degraded slowly and consequently inhibits replication. Nucleotide substitutions within the RNAase E cleavage sequence alter RNAI half-life and plasmid copy number, changing also the incompatibility phenotype. RNAI variants lacking the sequence cleaved by RNAase E are eliminated by growth rate-dependent degradation, resulting in growth-responsive control of plasmid replication and copy number.     

Three regions in the DNA of plasmid pLS1 show sequence-directed static bending.

Three regions showing abnormal electrophoretic mobility, which is an indication of the existence of bends in DNA, have been observed in the DNA of plasmid pLS1. These loci have been characterized by assays designed to detect sequence-directed bending in DNA (temperature-dependence migration and two dimensional electrophoresis). The first region (locus B-1) was located within a fragment that contains a proposed inhibitor countertranscribed RNA (RNAII). The second locus (B-2) contains the plasmid plus origin of replication and the third region (locus B-3) was located in the vicinity of a putative antisense RNA (RNAI) of unknown function. The centres of the first two bent DNA regions were located by circular permutation assays at nucleotides 882 (locus B-1) and 634 (locus B-2). The bend centre of locus B-1 was found to be upstream of the promoter for the putative antisense RNAII. The centre of curvature in locus B-2 was located in the vicinity of the putative promoter of the replication proteins RepA and RepB and of a sequence that has three 11-bp direct repeats. The DNA sequence at this region showed the existence of A.T tracts, with an internal repeat of 10 to 11 base pairs, for five helix turns. A complex curvature in the DNA of pLS1 at locus B-2 that may have a regulatory role in plasmid replication is postulated.     

Maintenance of pBR322-derived plasmids without functional RNAI

pBR322-derived plasmids that lack the bla gene and 40% of the gene for the replication inhibitor, RNAI, have been constructed. Since the RNAI gene totally overlaps with the gene for the replication primer, RNAII, this primer is similarly defective and also lacks its normal promoter. The primer is presumed to by synthesized either from the counter-tet promoter (plasmid pCL59) or from an inserted lacUV5 promoter (plasmid pCL59-65). Based mainly on the observation that the plasmid Rom protein, which normally assists in the RNAI/RNAII interaction, has no effect on the replication of the RNAI/RNAII-defective plasmids, we suggest that the defective RNAI is not functional while the defective RNAII primer, although less efficient, still allows plasmid replication. The defective plasmids are fully compatible with the intact parent plasmid, indicating that they do not share a common control of replication. In the absence of antibiotics, the bacteria lose the defective plasmid, beginning after 80 generations; under the same conditions, the parent plasmid is retained even after 140 generations. During exponential growth of their host, the number of defective plasmids in a culture increases exponentially with a doubling time either smaller or greater than that of the host cell growth, depending on the growth medium and, in the case of pCL59-65, on the presence or absence of lac inducer IPTG. As a result of these differences in host cell growth and plasmid replication, the plasmids are either gradually diluted out or their copy number continually increases. This shows that, without RNAI, plasmid replication is uncoupled from the host cell growth and not, as usual, adjusted to it. It also implies that the RNAI mechanism is the only means of replication control for ColE1-type plasmids that senses and adjusts the copy number; limiting host factors cannot provide a back-up control to stabilize copy numbers.     

Directed evolution of colE1 plasmid replication compatibility: a fast tractable tunable model for investigating biological orthogonality

Plasmids of the ColE1 family are among the most frequently used in molecular biology. They were adopted early for many biotechnology applications, and as models to study plasmid biology. Their mechanism of replication is well understood, involving specific interactions between a plasmid encoded sense-antisense gene pair (RNAI and RNAII). Due to such mechanism, two plasmids with the same origin cannot be stably maintained in cells—a process known as incompatibility. While mutations in RNAI and RNAII can make colE1 more compatible, there has been no systematic effort to engineer new compatible colE1 origins, which could bypass technical design constraints for multi-plasmid applications. Here, we show that by diversifying loop regions in RNAI (and RNAII), it is possible to select new viable colE1 origins compatible with the wild-type one. We demonstrate that sequence divergence is not sufficient to enable compatibility and pairwise interactions are not an accurate guide for higher order interactions. We identify potential principles to engineer plasmid copy number independently from other regulatory strategies and we propose plasmid compatibility as a tractable model to study biological orthogonality.     

Design of a microaerobically inducible replicon for high-yield plasmid DNA production

A pUC-derived replicon inducible by oxygen limitation was designed and tested in fed-batch cultures of Escherichia coli. It included the addition of a second inducible copy of rnaII, the positive replication control element. The rnaII gene was expressed from P_trc and cloned into pUC18 to test the hypothesis that the ratio of the positive control molecule RNAII to the negative control element, RNAI, was the determinant of plasmid copy number per chromosome (PCN). The construct was evaluated in several E. coli strains. Evaluations of the RNAII/RNAI ratio, PCN and plasmid yield normalized to biomass (Y_pDNA/X) were performed and the initial hypothesis was probed. Furthermore, in high cell-density cultures in shake flasks, an outstanding amount of 126 mg/L of plasmid was produced. The microaerobically inducible plasmid was obtained by cloning the rnaII gene under the control of the oxygen-responsive Vitreoscilla stercoraria hemoglobin promoter. For this plasmid, but not for pUC18, the RNAII/RNAI ratio, PCN and Y_pDNA/X efficiently increased after the shift to the microaerobic regime in fed-batch cultures in a 1 L bioreactor. The Y_pDNA/X of the inducible plasmid reached 12 mg/g at the end of the fed-batch but the original pUC18 only reached ca. 6 mg/g. The proposed plasmid is a valuable alternative for the operation and scale-up of plasmid DNA production processes in which mass transfer limitations will not represent an issue.     

Arginine-rich RNA binding domain and protein scaffold domain of RNase E are important for degradation of RNAI but not for that of the Rep mRNA of the ColE2 plasmid

Expression of the replication initiator protein (Rep) of the ColE2 plasmid is controlled by antisense RNA (RNAI). Therefore alterations in processes and/or rates of degradation of these two RNAs would affect the Rep expression. Here, we have shown that the arginine-rich RNA binding domain (ARRBD) of RNase E is important for the initial endoribonucleolytic cleavage of RNAI but dispensable for the endoribonucleolytic cleavages of the Rep mRNA. We have also shown that the protein scaffold domain of RNase E is important for successive exoribonucleolytic degradation of RNAI, suggesting involvement of RhlB, but dispensable for that of the Rep mRNA. Such differences in the initiation and successive steps of degradation between RNAI and the Rep mRNA might be important in determining their individual degradation efficiencies required for a quick response to the changes in the plasmid copy number.