PcnB is required for the rapid degradation of RNAI, the antisense RNA that controls the copy number of CoIE1‐related plasmids

Abstract

The replication of ColE1-related plasmids is controlled by an unstable antisense RNA, RNAI, which can interfere with the successful processing of the RNAII primer of replication. We show here that a host protein, PcnB, supports replication by promoting the decay of RNAI. In bacterial strains deleted for PcnB a stable, active form of RNAI, RNAI*, which appears to be identical to the product of 5'-end processing by RNAase E, accumulates. This leads to a reduction in plasmid copy number. We show, using a GST-PcnB fusion protein, that PcnB does not interfere with RNAI/RNAII binding in vitro. The fusion protein, like PcnB, has polyadenylating activity and is able to polyadenylate RNAI (and also another antisense RNA, CopA) in vitro.

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

“…The decay of SL-E appears to be less dependent on PcnB than is that of RNA I -5 . This is correlated with the observation that RNA I -5 (whose stability is increased by pcnB mutation) is nearly absent (< 5%; He et al, 1993;Xu et al, 1993) from pcnB þ cells, while SL-E makes up 25-50% of total CopA (see above; Wagner et al, 1992). Thus, SL-E, although rapidly generated from CopA-FL, does not subsequently disappear as quickly as does RNA I -5 .…”

“…The more pronounced effect of PcnB deficiency on ColE1-like plasmids (10-fold reduction) has been shown to result from an Ϸ10-fold stabilization of the RNase E-cleaved RNA I species, RNA I -5 Xu et al, 1993). This shorter species is inhibition competent (Lin-Chao and Cohen, 1991;He et al, 1993). The results shown in Fig.…”

“…4, corrected for loading), is in good agreement with this expectation. The reason why copy number varies with the stability of the inhibitor is that (see also He et al, 1993) increasing the half-life of an inhibiting antisense RNA (by, e.g. 10-fold) will decrease the probability of translation of a nascent target mRNA (and a subsequent replication event) by a similar factor (Nordströ m and Wagner, 1994).…”

“…RNA preparations were performed as described by Blomberg et al (1990). Northern blot analysis was performed essentially as described by He et al (1993). Total RNA was separated on 6% polyacrylamide gels containing 7 M urea and subsequently transferred to GeneScreenPlus nylon membranes (DuPont) using a Bio-Rad Transblot Cell (20 V overnight).…”

“…The pcnB mutation also affects the copy number of the IncFII plasmid R1, although to a lesser extent (2-to 3-fold reduction; Masters et al, 1993) than it does that of ColE1 (Ϸ10-fold reduction; He et al, 1993;Masters et al, 1993). 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).…”