Control of ColE1 plasmid replication by antisense RNA

One of the two major classes of regulatory strategies that control plasmid copy number involves recognition via base pairing between two plasmid-encoded complementary RNAs. The detailed analysis of this control circuitry has revealed some features of regulatory mechanisms based on RNA-RNA interaction that distinguish them from those based on protein-nucleic acid interaction. These features provide a framework with which to understand other regulatory mechanisms based on RNA-RNA interaction, and will aid in the design of efficient artificial antisense RNA systems.     

Control of ColE1 plasmid replication. Intermediates in the binding of RNA I and RNA II.

Replication of plasmid ColE1 is regulated by a plasmid-specified small RNA (RNA I). RNA I binds to the precursor (RNA II) of the primer for DNA synthesis and inhibits primer formation. The process of binding of RNA I to RNA II that results in formation of a stably bound complex consists of a series of reactions forming complexes differing in the stability. Formation of a very unstable early intermediate that was previously inferred from the inhibition of stable binding caused by a second RNA I species was firmly established by more extensive studies. This complex is converted to a more stable yet reversible complex that was identified by its RNase sensitivity, which was altered from that of the earlier complex or from that of free RNA I or RNA II. In these complexes, most loops of RNA II interact with their complementary loops of RNA I. The kinetic and structural analyses of the binding process predict formation of a complex interacting at a single pair of complementary loops that precedes formation of these complexes. Thus the process of binding of RNA I to RNA II is seen to consist of a sequence of reactions producing a series of progressively more stable intermediates leading to the final product.     

Effect of altered efficiency of the RNA I and RNA II promoters on in vivo replication of ColE1-like plasmids in Escherichia coli

Summary Thermal inactivation of the dnaA gene product leads to a considerable decrease in the rate of replication of ColE1-like plasmids. To test the possiblity that the dnaA protein may affect synthesis of RNA I, which is an inhibitor of primer formation, or synthesis of RNA II, which is the primer precursor for replication of ColE1 (Tomizawa and Itoh 1982), the effect of the dnaA46 mutation on the efficiency of the RNA I and the RNA II promoters was examined. It appears that thermal inactivation of the dnaA protein results in a considerable increase in the activity of the RNA I promoter. We suggest that overproduction of RNA I in dnaA mutants grown at the restrictive temperature is responsible for the reduced replication of ColE1-like plasmids.It has been found that addition of rifampicin to cultures of the dnaA46 or the dna ⁺ strain grown at 42°C results in a dramatic increase in the rate of replication of ColE1-like plasmids. We show that the activity of the RNA II promoter at 42°C is exceptionally resistant to rifampicin. In the presence of the drug, this leads, to an altered ratio of RNA I to RNA II, in favor of the latter RNA species.     

Processing of plasmid DNA with ColE1-like replication origin

With the increasing utilization of plasmid DNA as a biopharmaceutical drug, there is a rapidly growing need for high quality plasmid DNA for drug applications. Although there are several different kinds of replication origins, ColE1-like replication origin is the most extensively used origin in biotechnology. This review addresses problems in upstream and downstream processing of plasmid DNA with ColE1-like origin as drug applications. In upstream processing of plasmid DNA, regulation of replication of ColE1-like origin was discussed. In downstream processing of plasmid DNA, we analyzed simple, robust, and scalable methods, which can be used in the efficient production of pharmaceutical-grade plasmid DNA.     

Characterization of the Structure and Melting Behavior of the Loop I fragment of ColE1 RNA I

Abstract

We have synthesized two RNA fragments: a 42-mer corresponding to the full loop I sequence of the loop I region of ColE1 antisense RNA (RNA I), plus three additional Gs at the 5′-end, and a 31-mer which has 11 5′-end nucleotides (G(-2)-U9) deleted. The secondary structure of the 42-mer, deduced from one- and two-dimensional NMR spectra, consists of a stem of 11 base-pairs which contains a U-U base-pair and a bulged C base, a 7 nucleotide loop, and a single-stranded 5′ end of 12 nucleotides. The UV-melting study of the 42-mer further revealed a multi-step melting behavior with transition temperatures 32°C and 71°C clearly discernible. In conjunction with NMR melting study the major transition at 71°C is assigned to the overall melting of the stem region and the 32°C transition is assigned to the opening of the loop region. The deduced secondary structure agrees with that proposed for the intact RNA I and provides structural bases for understanding the specificity of RNase E.     

Characterization of a promoter up mutation in the -35 region of the promoter of the primer for ColE1 replication

Summary A point mutation in the -35 region of the promoter of the primer for initiation of DNA replication in the plasmid pMB1 was characterized. This base change causes a promoter up phenotype. The analysis of a second mutant obtained by site-directed mutagenesis allowed the exclusion of a role in the phenotype for the potential intrastrand secondary structure as well as for the methylation state of the DNA in the promoter region. The promoter up phenotype is concluded to be due to a change in the primary structure of the — 35 element with the consequent production of a better cluster of hydrogen bond donors and acceptors for the RNA polymerase.     

Interaction between RNA1 and the primer precursor in the regulation of Co1E1 replication

The analysis of a large number of independent mutants in the target of one of the inhibitors of pMB1 replication suggests that RNA1 regulates primer formation by base-pairing with the complementary sequence in the primer precursor. We conclude that the number of bases that are involved in the hydrogen bonding responsible for the specificity of the mechanism that controls plasmid replication and incompatibility properties is not much larger than seven. Five of these bases are located in the central loop and two in loop I of the RNA primer cloverleaf structure. Twenty-two single, double or triple mutants, with different nucleotide sequences in these seven bases, maintain an active mechanism of control, though with altered specificity. The efficiency of the inhibition mechanism correlates with the delta G value of the hydrogen bonds between the nucleotides of the two heptamers postulated to be involved in the interaction. The implications of these findings are discussed, and a molecular model of the interaction between RNA1 and the primer precursor is presented.