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

Abstract

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...

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

“…However, stabilization of the upper stem does not account for the extremely low level of expression of repA with the SLI.LM⌬, suggesting that other factors such as the size of the SLI loop or the position of the proximal pseudoknot bases within this loop may be important for efficient formation of the pseudoknot. This notion is supported by data showing that when C587 of SLI.LM is replaced by a G so that the hairpin loop of SLI.LM becomes identical to that of the other SLIs tested here, the level of repA expression is approximately 16-fold higher than that seen with SLI.LM⌬, the mutant in which C587 had been deleted (4).…”

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).

“…Similarly, compatibility between members of the extended RepFIIA family is also a consequence of sequence variation in both loop and upper stem sequences in CopA (Athanasopoulos et al, 1995). Given the variation between stem sequences of CopA, from the other IncFII-related replicons shown in Fig.…”

“…The replicons from plasmids belonging to IncB, IncFII, IncFIC, IncI " , IncK, IncL\M and IncZ have been shown to share a common replication control mechanism, namely antisense control, and to share some homology between both DNA and protein sequences (Athanasopoulos et al, 1995 ;Hama et al, 1990 ;Praszkier et al, 1991 ;Saadi et al, 1987). In the IncFII replicon of R1 an antisense RNA molecule (CopA) inhibits synthesis of the replication protein (RepA) by binding to the leader region of the repA mRNA (CopT).…”

Pseudoknot-Dependent Translational Coupling inrepBAGenes of the IncB Plasmid pMU720 Involves Reinitiation

“…(10,11). However, pMU604 is only distantly related to the IncB and IncI 1 plasmids, so its nucleotide sequence, including the region encoding elements that regulate expression of repA, is significantly different from that of the other two plasmids (10). Pseudoknots are involved in both inhibition and activation of initiation of translation in eubacteria.…”

“…Diagram d shows that the interaction of RNAI with SLI preempts formation of the pseudoknot by sequestering the proximal pseudoknot bases; it also indirectly inhibits the formation of the pseudoknot by hindering the access of ribosomes to the TIR of repB. (10,11). However, pMU604 is only distantly related to the IncB and IncI 1 plasmids, so its nucleotide sequence, including the region encoding elements that regulate expression of repA, is significantly different from that of the other two plasmids (10).…”