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

Wagner, E. Gerhart H.; Blomberg, P.; Nordström, Kurt

doi:10.1002/j.1460-2075.1992.tb05160.x

Cited by 46 publications

(42 citation statements)

References 34 publications

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“…The decay of SL-E appears to be less dependent on PcnB than is that of RNA I -5 . This is correlated with the observation that RNA I -5 (whose stability is increased by pcnB mutation) is nearly absent (< 5%; He et al, 1993;Xu et al, 1993) from pcnB þ cells, while SL-E makes up 25-50% of total CopA (see above; Wagner et al, 1992). Thus, SL-E, although rapidly generated from CopA-FL, does not subsequently disappear as quickly as does RNA I -5 .…”

Section: Discussionsupporting

confidence: 72%

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

Söderbom

Binnie

Masters

et al. 1997

Molecular Microbiology

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SummaryThe 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 3Ј 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 3Ј 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.

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Section: Discussionsupporting

confidence: 72%

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

Söderbom

Binnie

Masters

et al. 1997

Molecular Microbiology

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“…This concept was initially proposed by for the interaction of RNAI and RNAII of ColEl and was later supported by both in vivo and in vitro studies of the interaction of the CopA antisense RNA of plasmid R1 with its target CopT (31)(32)(33). In vitro experiments with the R1 system even suggested that formation of the kissing complex is sufficient for the inhibitory action of CopA to occur (43). These results stress the critical role of single-stranded loops within antisense RNA molecules for their regulatory function.…”

Section: Materlils and Methodsmentioning

confidence: 89%

RepR protein expression on plasmid pIP501 is controlled by an antisense RNA-mediated transcription attenuation mechanism

Brantl¹,

Birch-Hirschfeld²,

Behnke³

1993

J Bacteriol

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Expression of the rate-limiting initiator protein RepR of plasmid pIP501 is controlled by the antisense RNAIII. Mutational alteration of individual G residues within the single-stranded loops of RNAIII led to an increase in copy number. In contrast to the G-rich single-stranded loops, two smaller AT-rich loops of RNAIII were found to be dispensable for its inhibitory function. Reciprocal mutations in the same loop compensated for each other's effect, and a destabilization of the major stem structure of RNAIII also resulted in an increased copy number. These data were consistent with the idea that the interaction of RNAIII with its target starts with the formation of a kissing complex between the single-stranded loops of both molecules. The repR mRNA leader sequence, which includes the target of RNAIIIs is able to assume two alternative structures due to the presence of two inverted repeats the individual sequences of which are mutually complementary. In the presence of the antisense RNAIII, one of these inverted repeats (IR2) is forced to fold into a transcriptional terminator structure that prevents transcription of the repR gene. In the absence of RNAIII, formation of the transcriptional terminator is prevented and expression of the essential repR gene can proceed normally. This antisense RNA-driven transcriptional attenuation mechanism was supported by extensive deletional analysis and direct evidence that IR2 functions as a transcriptional terminator.

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“…RNA-E binds to and forms a duplex with its complementary sequence in the repAl mRNA (4,18,37,41). The binding of RNA-E to repAl mRNA inhibits translation of a 24-amino-acid leader peptide, whose translation promotes that of repAl (3,45).…”

Section: Discussionmentioning

confidence: 99%

Suppression of replication-deficient mutants of IncFII plasmid NR1 can occur by two different mechanisms that increase expression of the repA1 gene

Wang

Womble

et al. 1993

J Bacteriol

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Replication-proficient (Rep') revertants were isolated from mutants of IncFll plasmid NR1 that were replication defective (Rep-). The parental Rep-plasmids contained a mutation that inactivated promoter PE for transcription of RNA-E, a trans-acting repressor of translation of the essential RepAl replication initiation protein of NR1. The PE mutation also introduced a nonsense codon into a leader peptide gene that precedes and slightly overlaps the repAl translation initiation site in the mRNA. This reduced the rate of synthesis of RepAl by uncoupling its translation from that of the leader peptide. The reduced rate of RepAl synthesis was responsible for the Rep-phenotype. All Rep' revertants retained the PE mutation and contained second-site mutations responsible for suppression of the Rep-phenotype. One Rep+ revertant contained a second mutation adjacent to the Shine-Dalgarno sequence of repAl. Another Rep+ revertant contained a mutation in the repA2 gene, which encodes the trans-acting repressor of transcription of repAl. By using translational JacZ gene fusions, it was found that both kinds of suppressor mutation increased the expression of repAl to a level sufficient to support replication. In both cases, the synthesis of RepAl remained uncoupled from that of the leader peptide. The Shine-Dalgarno mutation increased the rate of leader peptide-independent translation of repAl mRNA and also reduced the sensitivity of repAl mRNA to inhibition by RNA-E. The repA2 mutation inactivated the RepA2 repressor and increased the rate of transcription ofrepAl mRNA. The translational lacZ gene fusions were used to assess the range of regulation of expression of repAl provided by each of the RNA-E and RepA2 regulatory circuits. By constructing miniplasmids that contained various combinations of the mutations, the contributions of the RNA-E and RepA2 regulatory circuits were assessed with respect to control of plasmid copy number and stable inheritance. Plasmids that lacked either circuit were less stable than wild-type plasmids.

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

Cited by 46 publications

References 34 publications

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

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

RepR protein expression on plasmid pIP501 is controlled by an antisense RNA-mediated transcription attenuation mechanism

Suppression of replication-deficient mutants of IncFII plasmid NR1 can occur by two different mechanisms that increase expression of the repA1 gene

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