Role of countertranscript RNA in the copy number control system of an IncB miniplasmid

Praszkier, Judyta; Bird, Phillip I.; Nikoletti, S; Pittard, J

doi:10.1128/jb.171.9.5056-5064.1989

Cited by 38 publications

(46 citation statements)

References 55 publications

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“…2, and the effect on repA expression was assayed, using low-copy-number plasmids (approximately one or two copies per chromosome) with translational fusions in which codon 23 of repA was fused in phase with codon 8 of lacZ. The effects of the various mutations on the regulation of repA by RNAI were determined by performing assays with either the gene for RNAI (producing saturating RNAI levels) or the gene for the RNA complementary to RNAI (i.e., "target" RNA to titrate out RNAI) in trans on a multicopynumber plasmid (-20 to 30 copies per chromosome) (14,15).…”

Section: Resultsmentioning

confidence: 99%

“…Vector (pBR322) or its derivatives were present in trans. RNAI (pMU617) carries nt 438 to 718 of pMU720 and therefore expresses RNAI but not RNAII (14). Target (pMU662) carries nt 1 to 637 of pMU720 (14) and thus expresses the leader sequence of RNAII (stem-loop I), which is complementary to RNAI, but does not express RNAI.…”

Section: Discussionmentioning

confidence: 99%

“…pBR322 derivatives. The construction of these plasmids has been described previously (14). pMU617, which is pBR322 carrying nt 438 to 718 of pMU720 (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…The replication frequency of the B group miniplasmid pMU720 is thought to be dependent on the expression of the repA gene, which is negatively regulated primarily at the posttranscriptional level by a small countertranscript RNA, RNAI (14,15). RNAI is transcribed from the opposite strand of, and is complementary to, the leader region of the mRNA coding for repA (RNAII) (see Fig.…”

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Mutations affecting pseudoknot control of the replication of B group plasmids

1993

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The translational initiation region of the mRNA for the replication initiation protein (RepA) of pMU720 is predicted to be sequestered in an inhibitory secondary structure designated stem-loop III. Activation of repA translation requires both the disruption of stem-loop III by ribosomes involved in the translation and termination of the leader peptide RepB and the formation of a pseudoknot, a tertiary RNA structure.Disruption of stem-loop III by site-directed mutagenesis was found to be insufficient to allow high repA expression in the absence of pseudoknot formation, indicating that the pseudoknot acts as an enhancer of repA translation. Furthermore, extending the length of the leader peptide RepB and changing the distance between the pseudoknot and repA Shine-Dalgarno sequence were found to have major elfects on the translation of repA.Plasmid replication in prokaryotes is, in many cases, dependent on a replication initiation protein (Rep), whose expression determines a plasmid's copy number and stability. In the case of pT181 and IncFII plasmids, the regulators of Rep synthesis are small countertranscript RNAs which inhibit Rep expression by binding with their target RNAs. In pT181, binding of the countertranscript RNA to the mRNA for the Rep protein is proposed to cause premature transcriptional termination by altering the folding of the RNA (9, 13). The mechanism by which the countertranscript RNAs of the IncFII plasmids Rl and NR1 regulate Rep expression is not clear. However, it is thought that they indirectly regulate Rep expression by sterically inhibiting the translation of a leader peptide. Translation of the Rep protein is believed to be dependent on the translation of the leader peptide, and the two genes are said to be translationally coupled (4, 23).The replication frequency of the B group miniplasmid pMU720 is thought to be dependent on the expression of the repA gene, which is negatively regulated primarily at the posttranscriptional level by a small countertranscript RNA, RNAI (14,15). RNAI is transcribed from the opposite strand of, and is complementary to, the leader region of the mRNA coding for repA (RNAII) (see Fig. 1). Computer analysis of the folding of RNAII indicates that the translational initiation region (TIR) of repA is sequestered within a secondary structure designated stem-loop III (see Fig. 1 and Fig. 2). It is postulated that stem-loop III inhibits ribosome access to the repA TIR. Previous studies have revealed that for repA to be expressed, stem-loop III must be disrupted by the translation and termination of a small leader peptide RepB, and a pseudoknot has to form (15). Pseudoknot formation is essential for the translation of repA and involves pairing between complementary sequences in RNAII. One of these sequences lies in the loop of a large structure called stem-loop I (proximal pseudoknot sequence), which is complementary to RNAI (see Fig. 2), and the other involves bases adjacent to the ShineDalgarno (SD) sequence of repA (distal pseudoknot sequence). RNAI is thought to...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…pBR322 derivatives. The construction of these plasmids has been described previously (14). pMU617, which is pBR322 carrying nt 438 to 718 of pMU720 (Fig.…”

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Mutations affecting pseudoknot control of the replication of B group plasmids

1993

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“…For example, replication of the ColE1 plasmids is regulated by an antisense RNA, which by binding to a preprimer RNA alters its folding and prevents its subsequent processing to form a primer for the initiation of DNA synthesis (19,24,49,50). The antisense RNAs of the pT181, FII, IncI 1 and IncB plasmids inhibit the expression of the genes coding for the essential replication initiation proteins (Rep) (13,30,31,35,42,46). In pT181, binding of the antisense alters the folding of the rep mRNA, resulting in transcriptional attenuation of the message (31).…”

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confidence: 99%

The replication of an IncL/M plasmid is subject to antisense control

1995

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A 2,385-bp sequence that contains the information for the autonomous replication of the IncL/M plasmid pMU604 was characterized. Genetic analyses revealed that the replicon specifies at least four structural genes, designated repA, repB, repC, and rnaI. The repA gene encodes a protein with a molecular weight of 40,861 which probably functions as an initiator for replication. The functions of the proteins of the repB and repC genes are unclear; however, mutations in the start codon of repB reduced the expression of both repB and repA, indicating that these two genes are translationally coupled. The rnaI gene encodes a small antisense RNA of about 75 to 77 bases and is responsible for the incompatibility phenotype, thus implicating its role as the main copy number determinant. RNAI exerts its effect in trans to repress the expression of repA at the posttranscriptional level. Furthermore, two complementary sequences of 8 bases, with the potential to interact and form a putative pseudoknot structure, were identified in the leader region of the repA mRNA. Base-pairing between the two complementary sequences was shown to be critical for efficient repA expression. A model for the regulation of pMU604 replication involving both translational coupling and pseudoknot formation is proposed.Plasmids that employ a system of copy number control that involves inhibitor molecules binding to target sequences generally regulate their replication via a small antisense RNA molecule. The target of this inhibitory RNA is the complementary region of a large RNA transcript, whose product is essential for replication. Both antisense and target RNAs are highly structured molecules capable of assuming stable stem-andloop configurations. These structural motifs are just as important as their primary sequences for efficient and specific interaction between antisense and target molecules (6,10,32,34,43,47,52). Because the small RNAs act in trans, they are also able to affect the replication of other plasmids showing the same specificity of replication control. The ability to interact with the replication machinery of other plasmids means that antisense RNAs act as incompatibility determinants (29).Although a number of plasmids use antisense RNA to regulate the frequency with which they initiate replication, the particular molecular mechanism differs for each system. For example, replication of the ColE1 plasmids is regulated by an antisense RNA, which by binding to a preprimer RNA alters its folding and prevents its subsequent processing to form a primer for the initiation of DNA synthesis (19,24,49,50). The antisense RNAs of the pT181, FII, IncI 1 and IncB plasmids inhibit the expression of the genes coding for the essential replication initiation proteins (Rep) (13,30,31,35,42,46). In pT181, binding of the antisense alters the folding of the rep mRNA, resulting in transcriptional attenuation of the message (31). Control of copy number in the FII plasmids R1 and NR1 involves the hybridization of the antisense RNA with its complementary sequ...

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Control of replication of plasmid R1: structures and sequences of the antisense RNA, CopA, required for its binding to the target RNA, CopT.

1990

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The replication frequency of plasmid R1 is determined by the availability of the RepA protein, which acts at the origin of replication to promote initiation. Synthesis of RepA is negatively regulated both at the transcriptional and post‐transcriptional levels. Post‐transcriptional control is exerted through the action of an antisense RNA, CopA RNA. The target of CopA RNA, CopT RNA, is located in the leader region of the RepA mRNA. Binding between CopA and CopT inhibits repA expression. We have previously presented an in vitro analysis of the binding reaction between CopA and CopT RNAs. In this communication, we extend the in vitro analysis by determining the regions of CopA required for binding, and also demonstrate that binding occurs in at least two steps. The first step is the formation of an initial, transient complex; stem‐loop II is the structure in CopA necessary and sufficient for this step. The subsequent step(s), resulting in the formation of a complete duplex, requires a stretch of single‐stranded nucleotides located 5′ to stem‐loop II in CopA, and its counterpart in CopT. We show that the single‐stranded region can be positioned on either side of stem‐loop II provided that there is a complementary stretch of nucleotides in CopT, indicating that the second step(s) is not sequence‐specific. Furthermore, the effects of salt concentration and temperature on the binding reaction indicate that duplex formation occurs through a mechanism of gradual intra‐strand breaking and inter‐strand formation of hydrogen bonds.

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Role of countertranscript RNA in the copy number control system of an IncB miniplasmid

Cited by 38 publications

References 55 publications

Mutations affecting pseudoknot control of the replication of B group plasmids

Mutations affecting pseudoknot control of the replication of B group plasmids

The replication of an IncL/M plasmid is subject to antisense control

Control of replication of plasmid R1: structures and sequences of the antisense RNA, CopA, required for its binding to the target RNA, CopT.

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