Replication control in plasmid R1: duplex formation between the antisense RNA, CopA, and its target, CopT, is not required for inhibition of RepA synthesis.

The replication frequency of plasmid R1 is regulated by an antisense RNA, CopA, which inhibits the synthesis of the rate-limiting initiator protein RepA. The inhibition requires an interaction between the antisense RNA and its target, CopT, in the leader of the RepA mRNA. This binding reaction has previously been studied in vitro, and the formation of a complete RNA duplex between the two RNAs has been demonstrated in vitro and in vivo. Here we investigate whether complete duplex formation is required for CopA-mediated inhibition in vivo. A mutated copA gene was constructed, encoding a truncated CopA which is impaired in its ability to form a complete CopA/CopT duplex, but which forms a primary binding intermediate (the 'kissing complex'). The mutated CopA species (S-CopA) mediated incompatibility against wild-type R1 plasmids and inhibited RepA-LacZ fusion protein synthesis. Northern blot, primer extension and S1 analyses indicated that S-CopA did not form a complete duplex with CopT in vivo since bands corresponding to RNase III cleavage products were missing. An in vitro analysis supported the same conclusion. These data suggest that formation of the 'kissing complex' suffices to inhibit RepA synthesis, and that complete CopA/CopT duplex formation is not required. The implications of these findings are discussed.     

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.     

P1 plasmid replication. Purification and DNA-binding activity of the replication protein RepA

The minimal P1 replicon encompasses an open reading frame for the essential replication protein, RepA, bracketed by two sets of multiple 19-base pair repeated sequences, incA and incC. This study focused on the interaction of RepA with the incC and incA repeated sequences because earlier studies suggested that incA might control P1 copy number by titrating limiting amounts of RepA and because the incC repeats, which are part of the origin of replication, contain the promoter for repA. RepA is essential for origin function, autoregulates its own synthesis from the promoter, and, when overproduced, blocks origin function. In this study, RepA was overproduced from an expression vector and purified to 90% homogeneity. The binding of RepA to the DNA encompassing repeat sequences was assayed by monitoring the mobility of protein-DNA complexes on polyacrylamide gels. Distinct species of retarded bands were seen with the maximum number of bands corresponding to the number of repeats present in the target fragment. No evidence was found for RepA binding to fragments not containing the repeats. This suggests that the specific binding of RepA to the repeats may be involved in each of the diverse activities of RepA.     

Role of the hepatitis C virus core+1 open reading frame and core cis-acting RNA elements in viral RNA translation and replication

Four conserved RNA stem-loop structures designated SL47, SL87, SL248, and SL443 have been predicted in the hepatitis C virus (HCV) core encoding region. Moreover, alternative translation products have been detected from a reading frame overlapping the core gene (core+1/ARFP/F). To study the importance of the core+1 frame and core-RNA structures for HCV replication in cell culture and in vivo, a panel of core gene silent mutations predicted to abolish core+1 translation and affecting core-RNA stem-loops were introduced into infectious-HCV genomes of the isolate JFH1. A mutation disrupting translation of all known forms of core+1 and affecting SL248 did not alter virus production in Huh7 cells and in mice xenografted with human liver tissue. However, a combination of mutations affecting core+1 at multiple codons and at the same time, SL47, SL87, and SL248, delayed RNA replication kinetics and substantially reduced virus titers. The in vivo infectivity of this mutant was impaired, and in virus genomes recovered from inoculated mice, SL87 was restored by reversion and pseudoreversion. Mutations disrupting the integrity of this stem-loop, as well as that of SL47, were detrimental for virus viability, whereas mutations disrupting SL248 and SL443 had no effect. This phenotype was not due to impaired RNA stability but to reduced RNA translation. Thus, SL47 and SL87 are important RNA elements contributing to HCV genome translation and robust replication in cell culture and in vivo.     

Control of replication of plasmid R1: the intergenic region between copA and repA modulates the level of expression of repA

The RepA protein of plasmid R1 is rate-limiting for initiation of R1 replication. Its synthesis is mainly regulated by interactions of the antisense RNA, CopA, with the leader region of the RepA mRNA, CopT. This work describes the characterization of several mutants with sequence alterations in the intergenic region between the copA gene and the repA reading frame. The analysis showed that most of the mutations led both to a decrease in stability of maintenance of mini-R1 derivatives and to lowered repA expression assayed in translational repA-lacZ fusion constructs. Destruction of the copA gene and replacement of the upstream region by the tac promoter in the latter constructs indicated that these mutations per se alter the expression of repA. In addition, we show that particular mutations in this region can directly affect CopA-mediated control, either by changing the kinetics of interaction of CopA RNA with the RepA mRNA and/or by modifying the activity of the copA promoter. These data indicate the importance of the region analysed in the process that controls R1 replication.     

Control of replication and segregation of R plasmid Rts1.

A mutant plasmid, pTW2, which was derived from the integrated Rst1 genome in the Escherichia coli chromosome, was studied as to its mode of replication at 30 degrees C. When Proteus mirabilis Pm17 harboring pTW2 was grown in broth at 30 degrees C, a considerable number of R- segregants (approximately 40%) were consistently observed. This indicates that pTW2 is unstable even at the permissive temperature for the replication of Rts1. The pTW2+ cells in a culture were heterogeneous with respect to the level of kanamycin resistance, ranging from 500 to 4,000 mug of the drug per ml. The amount of pTW2 deoxyribonucleic acid (DNA) relative to the Pm17 chromosomal DNA was about fivefold as large as that of Rts1 DNA in an exponentially growing culture. In addition, pTW2 in P. mirabilis continued to replicate after the chromosome had ceased to replicate, which was shown in the study of the inhibition of protein synthesis. Contrary to pTW2, the parent plasmid Rts1 is highly stable, and the relative percent Rts1 DNA is maintained at approximately 7% in any cultural conditions at a permissive temperature. These results suggest that copies of pTW2 may not segregate evenly into the host progeny upon cell division and that the replication of pTW2 does not coordinate with that of the chromosome. A remarkable instability of pTW2 as well as an increase in the relative percent pTW2 DNA was also shown when E. coli were used as the host cells. These results suggest the possibility that there is a gene or a gene cluster on the Rst1 genome responsible for the control of both replication and segregation of Rts1.     

Importance of the C terminus of plasmid Rts1 RepA protein for replication and incompatibility of the plasmid.

RepA protein, essential for replication of plasmid Rts1, was found to bind in vivo immediately upstream of the repA promoter in studies with mini-Rts1 derivatives with deletions in the upstream region of repA. We constructed another series of repA mutants that would encode RepA derivatives containing oligopeptide substitutions in place of the carboxyl-terminal six amino acids. These modified RepA proteins could not activate ori (Rts1) at all and showed various degrees of incompatibility, or no incompatibility, toward a mini-Rts1 plasmid. These results suggest that the carboxyl-terminal six (or fewer) amino acids of RepA are important for exerting replication and incompatibility functions. One of the RepA derivatives, which showed an evident incompatibility without initiating replication, was examined for its ability to repress the repA gene.     

The incompatibility product of IncFII R plasmid NR1 controls gene expression in the plasmid replication region.

The incompatibility properties of IncFII R plasmid NR1 were compared with those of two of its copy number mutants, pRR12 and pRR21. pRR12 produced an altered incompatibility product and also had an altered incompatibility target site. The target site appeared to be located within the incompatibility gene, which is located more than 1,200 base pairs from the plasmid origin of replication. The incompatibility properties of pRR21 were indistinguishable from those of NR1. Lambda phages have been constructed which contain the incompatibility region of NR1 or of one of its copy mutants fused to the lacZ gene. In lysogens constructed with these phages, beta-galactosidase was produced under the control of a promoter located within the plasmid incompatibility region. Lysogens containing prophages with the incompatibility regions from pRR12 and pRR21 produced higher levels of beta-galactosidase than did lysogens containing prophages with the incompatibility region from the wild-type NR1. The introduction into these inc-lac lysogens of pBR322 plasmids carrying the incompatibility regions of the wild-type or mutant plasmids resulted in decreased levels of beta-galactosidase production. For a given lysogen, the decrease was greater when the pBR322 derivative expressed a stronger incompatibility toward the plasmid from which the fragment in the prophage was derived. This suggested that the incompatibility product acts on its target to repress gene expression in the plasmid replication region.     

Regulated translation termination at the upstream open reading frame in s-adenosylmethionine decarboxylase mRNA.

The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a cis-acting element that confers feedback control by cellular polyamines on translation of this message. Recent studies demonstrated that elevated polyamines inhibit synthesis of the peptide encoded by the uORF by stabilizing a ribosome paused in the vicinity of the termination codon. These studies suggested that polyamines act at the termination step of uORF translation. In this paper, we demonstrate that elevated polyamines stabilize an intermediate in the termination process, the complete nascent peptide linked to the tRNA that decodes the final codon. The peptidyl-tRNA molecule is found associated with the ribosome fraction, and decay of this molecule correlated with release of the paused ribosome from the message. Furthermore, the stability of this complex is influenced by the same parameters that influence regulation by the uORF in vivo, namely the concentration of polyamines and the sequence of the uORF-encoded peptide. These results suggest that the regulated step in uORF translation is after formation of the peptidyl-tRNA molecule but before hydrolysis of the peptidyl-tRNA bond. This regulation may involve an interaction between the peptide, polyamines, and a target in the translational apparatus.     

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.     

Effects of the open reading frames in the Rous sarcoma virus leader RNA on translation.

Three short open reading frames (ORFs) reside in the 5' leader of Rous sarcoma virus (RSV) and are conserved in all avian sarcoma-leukosis retroviruses. Both extensions of the lengths of the ORFs and alterations in their initiation codons affect viral replication and gene expression. To determine whether the effects on viral replication were due to translational regulation mediated by the ORFs, we examined translation following mutation of the initiation and termination codons of each of the three ORFs. We found that the ORFs marginally enhanced downstream gene expression. Moreover, repression of downstream gene translation was proportional to the lengths of the elongated ORFs and depended on the initiation contexts of the AUG codons. Although the ORFs play a major role in viral activities, their effects on translation were relatively minor. Rather, the ORFs may affect the fate of unspliced avian retroviral RNA in chronically infected cells by participating in the sorting of viral RNA for either translation or encapsidation into virions.     

Functional relationship between parts of the replication region of plasmid ColE1.

The inhibition of plasmid ColE1 replication caused by a deletion of the ColE1 plasmid replication origin has been previously reported (T. Hashimoto-Gotoh and J. Inselburg, J. Bacteriol. 139:597-619). Evidence is presented showing that restoration of the deleted nucleotide sequence in the precise relationship it normally has to the rest of the replication region is essential for restoration of ColE1 replication capability to the deletion mutant.