Control of replication of plasmid R1: the duplex between the antisense RNA, CopA, and its target, CopT, is processed specifically in vivo and in vitro by RNase III.

Abstract

The replication frequency of IncFH plasmids is regulated through the availability of a rate-limiting protein, RepA.

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

“…2B). The presence of this species is dependent on RNase III cleavage of CopA/target RNA duplexes and has been characterized extensively in previous work (Blomberg et al, 1990). The 5Ј segment of CopA resulting from RNase E cleavage has never been detected, suggesting that its degradation is extremely rapid.…”

“…The experiments were carried out as described previously (Blomberg et al, 1990; Experimental procedures). The results are shown as an autoradiogram and as a schematic drawing ( Fig.…”

“…Furthermore, as is the case with RNA I in ColE1-containing cells, two forms of CopA are found in R1-containing cells, a full-length form of Ϸ90 nt, CopA-FL, and an Ϸ65-nt-long 3Ј segment, SL-E, which is a cleavage product previously hypothesized to be generated by RNase E (Blomberg et al, 1990). We, therefore, sought to establish whether turnover of the antisense RNAs of these two unrelated plasmid groups occurs by similar pathways.…”

An unusual structure formed by antisense-target RNA binding involves an extended kissing complex with a four-way junction and a side-by-side helical alignment

“…The role of CopA is to regulate the synthesis of RepA protein+ Previous work showed that the stable CopA/ CopT complex detected in vitro prevents ribosome from initiation complex formation at the tap RBS (Malmgren et al+, 1996)+ CopI, the truncated antisense RNA unable to form fully paired duplexes, also repressed repA expression , and the extended kissing complex (CopI-CopT) sufficed to transiently interfere with ribosome binding (Malmgren et al+, 1996) (Öhman & Wagner, 1989;Malmgren et al+, 1997)+ The model proposed here for the CopI-CopT complex is a bulky structure (Fig+ 7) that might, by steric hindrance, prevent ribosome binding at tap+ In fulllength CopA, the presence of its 59 extension has several functional implications+ It stabilizes the extended kissing complex, providing approximately three helical turns of double-stranded RNA immediately 59 of the tap SD+ Thus, its main role is to promote complete and irreversible inhibition of tap translation+ This intermolecular helix is also a substrate for RNase III cleavage, although destabilization of repA mRNA contributes little to control of repA expression (Blomberg et al+, 1990)+ Finally, factors that alter CopA turnover will also affect plasmid copy number, because the degree of inhibition is correlated with the intracellular concentration of CopA+ The 59 tail of CopA carries a cleavage site for RNase E, the enzyme that initiates rapid turnover of CopA (Söder-bom et al+, 1997)+ In many regulatory antisense systems, the formation of complete antisense-target RNA duplexes appears to be a slow process in vitro and often becomes arrested at the stage of a stable binding intermediate (Wagner & Brantl, 1998;Zeiler & Simons, 1998)+ For CopA-CopT, topological barriers are encountered during helix propagation, especially at the four-way junction of the extended kissing complex+ Coaxial stacking with parallel packing of helices is known to be a general driving force towards RNA folding and probably contributes to the stabilization of the CopA-CopT binding intermediate+ Moreover, unwinding of the stems of CopA and CopT from helix C, although topologically possible, requires overcoming important energy barriers+ Thus, the CopA-CopT system appears to exploit binding intermediates as active key structures for the inhibitory step, rather than fully paired species+ So far, direct experimental evidence on structures of CopA-CopT complexes in bacterial cells is lacking+ However, identically located RNase III-dependent cleavages occurring on both RNAs in vitro and in vivo (Blomberg et al+, 1990;Malmgren et al+, 1997) provide circumstantial evidence that, even in the cell, binding may be arrested at the stage of the extended kissing complex stabilized by the intermolecular helix C+…”

“…CopT, CopA, CopI, and the mutant RNAs (H1-H3) were synthesized by T7 RNA polymerase from PCR-generated DNA fragments as described (Hjalt & Wagner, 1992)+ PCR fragments were generated from plasmid pGW58 (Blomberg et al+, 1990) carrying the wild-type copA/copT region+ Transcription of CopT yields a run-off product of 302 nt initiated with GG instead of the GU sequence of the wild-type repA mRNA+ The CopA RNA contains a 59 terminal G instead of an A residue+ Neither of these nucleotide changes affects structure or binding properties+ In CopA or CopI mutants, 2 or 3 bp were inverted as shown in Figure 6+ The complementary changes were also introduced in CopT mutants+ Purification of RNAs was performed by fast protein liquid chromatography (FPLC, Pharmacia) on a Bio-Sil TSK250 column+ The RNAs were eluted by 0+2 M sodium acetate, pH 6+5, containing 1% methanol, and precipitated+ 59-end labeling of dephosphorylated RNA was performed with T4 polynucleotide kinase and [g-32 P]ATP (Maniatis et al+,…”