Analysis of the incompatibility determinants of I-complex plasmids

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

doi:10.1128/jb.170.3.1311-1318.1988

Cited by 35 publications

(40 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These incompatibility data indicated that the IncZ antisense RNA is able to interact efficiently with the IncB RNAII and vice versa. The observation that the wild-type IncB RNAI could inhibit the expression of repA containing SLI.I 1 by 166-fold is also consistent with the incompatibility data, since incoming IncB plasmids displayed weak incompatibility against resident IncI 1 plasmids (18,22).…”

Section: Resultssupporting

confidence: 73%

Importance of structural differences between complementary RNA molecules to control of replication of an IncB plasmid

et al. 1997

Self Cite

View full text Add to dashboard Cite

Replication of the IncB miniplasmid pMU720 is dependent on the expression of repA, the gene encoding replication initiator protein RepA. Binding of a small antisense RNA (RNAI) to its complementary target (stem-loop I [SLI]) in the RepA mRNA prevents the participation of SLI in the formation of a pseudoknot that is an enhancer of translation of this mRNA. Thus, RNAI regulates the frequency of replication of pMU720 by controlling the efficiency of translation of the RepA mRNA. Mutational analysis of the two seven-base complementary sequences involved in formation of the pseudoknot showed that only the five central bases of each were critical for the formation of the pseudoknot. Physical analysis of SLI showed that despite the complete complementarity of its sequence to that of RNAI, the structures of the two molecules are different. The most prominent difference between the two structures is the presence of a 4-base internal loop immediately below the hairpin loop of SLI but not that of RNAI. Closure of this internal loop in SLI resulted in a 40-fold reduction in repA expression and loss of sensitivity of the residual expression to inhibition by RNAI. By contrast, repA expression was largely unaffected by the closure of a lower internal loop whose presence in SLI and RNAI is essential for effective interaction between these two molecules. These results suggest that the interaction of SLI with the distal pseudoknot bases is fundamentally different from the RNAI-SLI binding interaction and that the differences in structure between RNAI and SLI are necessary to allow SLI to be able to efficiently bind RNAI and to participate in pseudoknot formation.The miniplasmid pMU720 is a low-copy-number plasmid belonging to the incompatibility group B. The basic replicon consists of a 3.25-kb DNA fragment that contains the genetic information required for autonomous replication and copy number control. Replication of pMU720 requires the expression of the repA gene, whose product is thought to be essential and rate limiting for initiation of replication. Mutational and computer analyses of the folding of the repA mRNA (designated RNAII) indicate that its translation is normally inhibited by the presence of a secondary structure, stem-loop III (SLIII), which sequesters the repA translational initiation region (23,35). It has been established for both pMU720 (23, 35) and its close relative, the IncI 1 plasmid ColIb-P9 (1, 2), that expression of repA (designated repZ in ColIb-P9) requires activation of translation of the repA mRNA. This process involves the disruption of SLIII by the prior translation and correct termination of a small leader peptide (designated repB in pMU720 and repY in ColIb-P9), which enables the formation of a pseudoknot adjacent to the repA Shine-Dalgarno (SD) sequence. As first demonstrated in ColIb-P9, the formation of the pseudoknot is essential for the translation of repA and involves pairing between complementary sequences in the repA mRNA. The proximal pseudoknot sequence lies in the loop of a large sec...

show abstract

Section: Resultssupporting

confidence: 73%

Importance of structural differences between complementary RNA molecules to control of replication of an IncB plasmid

et al. 1997

Self Cite

View full text Add to dashboard Cite

show abstract

“…In pMU720, it has been shown that increasing the distance between the distal pseudoknot sequence and TIR of repA by as few as 2 bases drastically reduces the expression of this gene (54). The notion that these distances are important for function is supported by the observation that they have been conserved in all of the I-complex plasmids examined to date (13,28,36). On the other hand, the difference in the spacing between the distal pseudoknot sequence and the repB stop codon is less likely to be important, given that the corresponding codon of pMU720 could be moved a considerable distance with little effect on repA expression, as long as the ribosome terminating translation of repB was in a position to disrupt the secondary structure sequestering the distal pseudoknot sequence and TIR of repA without obstructing formation of the pseudoknot (37,53).…”

Section: Discussionsupporting

confidence: 51%

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

1995

Self Cite

View full text Add to dashboard Cite

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...

show abstract

“…This suggests that in both replicons, RepA synthesis is similarly regulated by CopA through inhibition of leader peptide translation. Mutations in both stem and loop regions have been shown to result in changes in incompatibility in both IncFII (Brady et al, 1983) and the IncI complex plasmids (Nikoletti et al, 1988) and variation in these regions is believed to be the basis for incompatibility in antisense-control-regulated replicons. Comparison of the predicted stem sequences of the five CopA molecules clearly shows variation in this region (Fig.…”

Section: Antisense Control Of Pgsh500 and Plv1402 Alpha Repliconsmentioning

confidence: 99%

Mosaic plasmids and mosaic replicons: evolutionary lessons from the analysis of genetic diversity in IncFII-related replicons The EMBL accession numbers for the sequences reported in this paper are AJ009980 (pGSH500 alpha replicon) and AJ009981 (pLV1402 alpha replicon).

et al. 2000

View full text Add to dashboard Cite

The alpha replicons of the multi-replicon plasmids pGSH500 and pLV1402 have been characterized by DNA sequence analysis. Analysis of the DNA sequence of a 3672 bp HindIII fragment from pFDT100, which contains the pGSH500 alpha replicon, revealed similarity to a number of replicons belonging to, or related to, those of the IncFII family. The replicon region contains copA, tapA, repA and oriR, and replication initiation and termination sites are related to those from the IncFII replicon of R1. A copB gene was found to lie upstream of the HindIII site in the parental plasmid pGSH500. Downstream of oriR, a 707 bp region shows 726 % identity to a region of the Escherichia coli chromosome at 433', suggesting this region of pGSH500 may have been incorporated into the plasmid during a past chromosomal recombination event. Oligonucleotide primers homologous to consensus regions in the copB and repA genes, and the oriR regions from a number of IncFII-related replicons were used to amplify replication regions from pLV1402. Analysis of the amplified regions has shown the presence of copB, copA, tapA and repA genes. Phylogenetic analysis of Rep protein sequences from the RepFIIA family of antisense-control-regulated replicons revealed the presence of three distinct subgroups of Rep proteins. Comparative analysis of DNA and protein sequences from members of the RepFIIA family provides evidence supporting the roles of both non-selective divergence in co-integrate (multi-replicon) plasmids and Chi-mediatedrecombination in replicon evolution, and in particular, that such processes may have been widespread in the evolution of the RepFIIA family.

show abstract

Analysis of the incompatibility determinants of I-complex plasmids

Cited by 35 publications

References 34 publications

Importance of structural differences between complementary RNA molecules to control of replication of an IncB plasmid

Importance of structural differences between complementary RNA molecules to control of replication of an IncB plasmid

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

Mosaic plasmids and mosaic replicons: evolutionary lessons from the analysis of genetic diversity in IncFII-related replicons The EMBL accession numbers for the sequences reported in this paper are AJ009980 (pGSH500 alpha replicon) and AJ009981 (pLV1402 alpha replicon).

Contact Info

Product

Resources

About