Suppression of ColE1 high-copy-number mutants by mutations in the polA gene of Escherichia coli.

We isolated three Escherichia coli suppressor strains that reduce the copy number of a mutant ColE1 high-copy-number plasmid. These mutations lower the copy number of the mutant plasmid in vivo up to 15-fold; the wild-type plasmid copy number is reduced by two- to threefold. The suppressor strains do not affect the copy numbers of non-ColE1-type plasmids tested, suggesting that their effects are specific for ColE1-type plasmids. Two of the suppressor strains show ColE1 allele-specific suppression; i.e., certain plasmid copy number mutations are suppressed more efficiently than others, suggesting specificity in the interaction between the suppressor gene product and plasmid replication component(s). All of the mutations were genetically mapped to the chromosomal polA gene, which encodes DNA polymerase I. The suppressor mutational changes were identified by DNA sequencing and found to alter single nucleotides in the region encoding the Klenow fragment of DNA polymerase I. Two mutations map in the DNA-binding cleft of the polymerase region and are suggested to affect specific interactions of the enzyme with the replication primer RNA encoded by the plasmid. The third suppressor alters a residue in the 3'-5' exonuclease domain of the enzyme. Implications for the interaction of DNA polymerase I with the ColE1 primer RNA are discussed.     

Origin-specific reduction of ColE1 plasmid copy number due to mutations in a distinct region of the Escherichia coli RNA polymerase

Mutations affecting a region of the Escherichia coli RNA polymerase have been isolated that specifically reduce the copy number of ColE1-type plasmids. The mutations, which result in a single amino acid alteration (G1161R) or a 41-amino acid deletion (Delta1149-1190) are located near the 3'-terminal region in the rpoC gene, which encodes the largest subunit (beta ') of the RNA polymerase. The rpoC deletion and the point mutation cause over 20- and 10-fold reductions, respectively, in the copy number of ColE1. ColE1 plasmid numbers are regulated by two plasmid-encoded RNAs: RNA II, which acts as a preprimer for the DNA polymerase I to start initiation of replication, and RNA I, its antisense inhibitor. Altered expression from the RNA I and RNA II promoters in vivo was observed in the RNA polymerase mutants. The RNA I/RNA II ratio is higher in the mutants than in the wild-type strain and this is most probably the main reason for the reduction in the ColE1 copy number in the two rpoC mutants.     

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.     

RNA1 is sufficient to mediate plasmid ColE1 incompatibility in vivo

The multicopy plasmid ColE1 specifies a small RNA designated RNA1 that has been implicated in copy number control and incompatibility. We have inserted a 148 base-pair ColE1 DNA fragment containing a promoter-less RNA1 gene into a plasmid vector downstream from the tryptophan promoter of Serratia marcesens. The ColE1 RNA1 produced by this plasmid is not functional in vivo due to the presence of 49 nucleotides appended to the 5′-terminus of the wild-type RNA1 sequence. Deletions of these sequences by Bal3I nuclease in vitro and genetic selection for ColE1 incompatibility function in vivo permitted isolation of a plasmid expressing wild-type ColE1 RNA1 initiated properly from the S. marcesens trp promoter. These experiments demonstrate that RNA1 is sufficient to mediate ColE1 incompatibility in vivo. In addition, several plasmids were isolated that contain altered RNA1 genes. These alterations consist of additions or deletions of sequences at the 5′-terminus of RNA1. Analysis of the ability of these altered RNA1 molecules to express incompatibility in vivo suggests that the 5′-terminal region of RNA1 is crucial for its function.     

Anatomy of the replication origin of plasmid ColE2-P9

The plasmid ColE2-P9 origin is a 32-bp region which is specifically recognized by the plasmid-specified Rep protein to initiate DNA replication. We analyzed the structural and functional organization of the ColE2 origin by using various derivatives carrying deletions and single-base-pair substitutions. The origin may be divided into three subregions: subregion I, which is important for stable binding of the Rep protein; subregion II, which is important for binding of the Rep protein and for initiation of DNA replication; and subregion III, which is important for DNA replication but apparently not for binding of the Rep protein. The Rep protein might recognize three specific DNA elements in subregions I and II. The relative transformation frequency of the autonomously replicating plasmids carrying deletions in subregion I is lower, and nevertheless the copy numbers of these plasmids in host bacteria are higher than those of the wild-type plasmid. Efficient and stable binding of the Rep protein to the origin might be important for the replication efficiency to be at the normal (low) level. Subregion II might be essential for interaction with the catalytic domain of the Rep protein for primer RNA synthesis. The 8-bp sequence across the border of subregions II and III, including the primer sequence, is conserved in the (putative) origins of many plasmids, the putative Rep proteins of which are related to the ColE2-P9 Rep protein. Subregion III might be required for a step that is necessary after Rep protein binding has taken place.     

Simulating RNA folding kinetics on approximated energy landscapes

We present a general computational approach to simulate RNA folding kinetics that can be used to extract population kinetics, folding rates and the formation of particular substructures that might be intermediates in the folding process. Simulating RNA folding kinetics can provide unique insight into RNA whose functions are dictated by folding kinetics and not always by nucleotide sequence or the structure of the lowest free-energy state. The method first builds an approximate map (or model) of the folding energy landscape from which the population kinetics are analyzed by solving the master equation on the map. We present results obtained using an analysis technique, map-based Monte Carlo simulation, which stochastically extracts folding pathways from the map. Our method compares favorably with other computational methods that begin with a comprehensive free-energy landscape, illustrating that the smaller, approximate map captures the major features of the complete energy landscape. As a result, our method scales to larger RNAs. For example, here we validate kinetics of RNA of more than 200 nucleotides. Our method accurately computes the kinetics-based functional rates of wild-type and mutant ColE1 RNAII and MS2 phage RNAs showing excellent agreement with experiment.     

Perturbation of the hierarchical folding of a large RNA by the destabilization of its Scaffold's tertiary structure

The P4-P6 domain serves as a scaffold against which the periphery and catalytic core organize and fold during Mg2+-mediated folding of the Tetrahymena thermophila ribozyme. The most prominent structural motif of the P4-P6 domain is the tetraloop-tetraloop receptor interaction which "clamps" the distal parts of its hairpin-like structure. Destabilization of the tertiary structure of the P4-P6 domain by perturbation of the tetraloop-tetraloop receptor interaction alters the Mg2+-mediated folding pathway. The folding hierarchy of P5c approximately P4-P6 > periphery > catalytic core that is a striking attribute of the folding of the wild-type RNA is abolished. The initial steps in folding of the mutant RNA are > or =50-fold faster than those of the wild-type ribozyme with the earliest observed tertiary contacts forming around regions known to specifically bind Mg2+. The interaction between the mutant tetraloop and the tetraloop receptor appears coincidently with slowly forming catalytic core tertiary contacts. Thus, the stability conferred upon the P4-P6 domain by the tetraloop-tetraloop receptor interaction dictates the preferred folding pathway by stabilizing an early intermediate. A sub-denaturing concentration of urea diminishes the early barrier to folding the wild-type ribozyme along with complex effects on the subsequent steps of folding the wild-type and mutant RNA.     

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.