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

Wu, Ruping; Wang, Xuesong; Womble, David D.; Rownd, Robert H.

doi:10.1128/jb.175.10.3161-3173.1993

Cited by 5 publications

(1 citation statement)

References 46 publications

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“…However, none of these measures to improve regulation of repB could be used in a low-copy-number plasmid such as pMU720, because all are predicted to reduce the plasmid's ability to correct downward fluctuations in its copy number, and unlike the IncFII plasmids, IncB plasmids do not contain a backup system to correct for downward fluctuations in copy number. In IncFII plasmids there is an additional promoter which is expressed only when the copy number of the plasmid reaches a critically low level (7,14,30), and derepression of this promoter has been shown to compensate for significantly decreased Rep expression in NR1 (32). Thus form and RepA cannot be synthesized.…”

Section: Resultsmentioning

confidence: 99%

Molecular analysis of RNAI control of repB translation in IncB plasmids

1994

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The translation of RepA, the replication initiation protein of the IncB plasmid pMU720, requires that its mRNA (RNAII) folds to form a pseudoknot immediately upstream of the repA Shine-Dalgarno sequence. The formation of this pseudoknot is dependent in turn on the translation and correct termination of a leader peptide, RepB. A small countertranscript RNA, RNAI, controls the replication of pMU720 by interacting with RNAII to negatively regulate the expression of repA both directly, by sequestering the proximal bases required for pseudoknot formation, and indirectly, by inhibiting the translation of repB. Inhibition of the translation of repB by RNAI was found to depend on the close proximity of the RNAI-RNAII complex to the translational initiation region of repB, indicating that the primary mechanism of RNAI control involves steric hindrance. Disruption of RNAI control of repB had only a small elfect on the copy number of the IncB plasmid, indicating that inhibition of the expression of repA by RNAI is achieved predominantly by inhibition of pseudoknot formation rather than by inhibition of repB translation.The IncB miniplasmid pMU720 is a low-copy-number plasmid with approximately two to four copies per cell. The basic replicon consists of a 3.25-kb DNA fragment which contains the genetic information required for autonomous replication and copy number control (Fig. 1). The replication frequency of pMU720 is dependent on the repA gene, the product of which is thought to be necessary for replication initiation (18,19). Expression of repA is negatively regulated at the posttranscriptional level by a small countertranscript RNA, RNAI, which is transcribed from the opposite strand to the RepA mRNA (RNAII) and is therefore complementary to it. Mutational and computer analyses of the folding of RNAII indicate that the translational initiation region (TIR) of repA is sequestered within a secondary structure, designated stem-loop III (SLIII), which prevents de novo translation by sterically inhibiting ribosome loading (20,29). It has been established for both the IncB plasmid pMU720 (20, 29) and its close relative, the IncI1 plasmid ColIb-P9 (1, 2), that for rep to be expressed, SLIII must be disrupted by the prior translation and correct termination of a small leader peptide, enabling the generation of a pseudoknot structure. As first demonstrated with ColIb-P9, the formation of the pseudoknot is essential for the translation of rep and involves pairing between complementary sequences in the Rep mRNA. The proximal pseudoknot sequence lies in the loop of stem-loop I (SLI), a large structure which is complementary to RNAI, and the distal pseudoknot sequence lies adjacent to the Shine-Dalgarno (SD) sequence of repA (Fig. 1). The pseudoknot, which must be in close proximity to the repA SD sequence, has been shown to act as an enhancer of the translation of repA. Its mode of action is unknown but may involve a direct pseudoknot-ribosome interaction (20,29).RNAI negatively regulates the translation of repA, primarily by pai...

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Section: Resultsmentioning

confidence: 99%

Molecular analysis of RNAI control of repB translation in IncB plasmids

1994

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Evolution of the Replication Regions of IncI and IncFII Plasmids by Exchanging Their Replication Control Systems

Kato

Mizobuchi

1994

DNA Research

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The basic replicons of bacterial plasmids consist of two sets of genetic systems, the replication-structural system and the replication control system. Comparison of nucleotide sequences suggested that the basic replicons of plasmids P307 (IncFI) and pMU2200 (IncZ) were generated by reciprocal recombination between ancestors of R100 (IncFII) and ColIb-P9 (IncI alpha), or vice versa. The plasmids of each pair, P307/pMU2200 and R100/ColIb-P9, are structurally unrelated to each other. Based on this information, we constructed in vitro and analyzed P307-like chimeric replicons from ColIb-P9 and R100. When the replication-structural region of ColIb-P9 was combined with the whole replication control region of R100, the resultant replicon replicated stably as R100 did. These results revealed that the basic replicons of the plasmids diverged by exchanging their replication control systems. Thus, we propose that the replication control systems of plasmids, in some cases, evolved independently of their structural systems, although these two systems work together to maintain the replication functions. We also showed that the reciprocal recombination was specified by the unique secondary structures of RNA involved in the control of expression of the genes encoding the replication initiator proteins.

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Replication control of plasmid R1: disruption of an inhibitory RNA structure that sequesters the repA ribosome‐binding site permits tap‐independent RepA synthesis

Blomberg

Engdahl

Malmgren

et al. 1994

Molecular Microbiology

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The replication frequency of plasmid R1 is controlled by an antisense RNA, CopA, that inhibits the synthesis of the replication initiator protein, RepA, at the post-transcriptional level. This inhibition is indirect and affects translation of a leader peptide reading frame (tap). Translation of tap is required for repA translation (Blomberg et al., 1992). Here we asked whether an RNA stem-loop sequestering the repA ribosome-binding site blocks tap translation-independent repA expression. Destabilization of this structure resulted in tap-independent RepA synthesis, concomitant with a loss of CopA-mediated inhibition; thus, CopA acts at the level of tap translation. Structure probing of RepA mRNAs confirmed that the introduced mutations induced a local destabilization in the repA ribosome-binding site stem-loop. An increased spacing between the repA Shine-Dalgarno region and the start codon permitted even higher repA expression. In Incl alpha/IncB plasmids, an RNA pseudoknot acts as an activator for rep translation. We suggest that the regulatory pathway in plasmid R1 does not involve an activator RNA pseudoknot.

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

Cited by 5 publications

References 46 publications

Molecular analysis of RNAI control of repB translation in IncB plasmids

Molecular analysis of RNAI control of repB translation in IncB plasmids

Evolution of the Replication Regions of IncI and IncFII Plasmids by Exchanging Their Replication Control Systems

Replication control of plasmid R1: disruption of an inhibitory RNA structure that sequesters the repA ribosome‐binding site permits tap‐independent RepA synthesis

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