from the negative controls contained cells with a normal cell morphology. Therefore, anti Sok PNA can inhibit Sok- mediated repression of hok mRNA. This result supports the model for direct Sok-RNA pairing with hok mRNA. Anti Sok PNAs cause mature hok mRNA accumulation According to the hok/sok model (20), blockage of Sok-RNA activity should lead to accumulation of the mature 30 end truncated hok mRNA. Therefore, if the model is correct and the anti Sok PNAs selectively sequester Sok-RNA, it should be possible to detect the truncated, mature isoform of hok mRNA in PNA-treated cells. Total RNA was prepared from hok/sok-carrying cells that were untreated or treated with rifampicin or anti Sok PNA at low micromolar concen- trations for 4 h. Northern analysis was performed using a probe that was complementary to a downstream region within the hok open reading frame (Figure 4A). The probe clearly detected pre-mRNA transcripts in all samples from hok/sok- carrying cells (Figure 4B). Importantly, in PNA-treated cells, the mature 30 end truncated form of hok mRNA was detected. The identity of the mature form of hok mRNA was indicated by comparison to rifampicin treated cells, which contain detectable amounts of mature hok mRNA following transcription arrest (20). Therefore, anti Sok PNA treatment sequesters free Sok-RNA and leads to accumula- tion of mature and active hok mRNA. Sequestered Sok molecules may be protected from 50 end degradation by Nucleic Acids Research, 2006, Vol. 34, No. 20 5919 RNase E, but it is unlikely that 30 end degradation by PNPase and RNase II is inhibited (31). PNA::Sok-RNA complex formation in vitro and interaction kinetics The phenotypic effects of anti Sok PNAs suggest that they are able to compete with the native interactions between hok mRNA and antisense Sok-RNA. If this is the case, it should be possible to determine the binding kinetics in vitro. Previ- ously, the binding-rate for Sok-RNA and hok mRNA interac- tions was determined in vitro by monitoring complex formation at low concentrations using gel shift analysis of radioactively labelled Sok-RNA (14). To determine the binding-rate for anti Sok PNAs, we ﬁrst re-established the hok mRNA::Sok-RNA interaction assay. Using 32P-labelled Sok-RNA and an excess of the mature form of hok mRNA we observed a clear reduction in free Sok-RNA and an increase in a slower migrating hok mRNA::Sok-RNA complex over time using PAGE (Figure 5A). By quantifying the reduction in free Sok-RNA we calculated the second- order binding-rate constant (k2) to be 1.4 · 105 M 1 s 1 (37 C) (Figure 5B), a value that is similar to that reported previously (14). In the same way, we determined the binding- rate of PNA::Sok-RNA complexes. Again, there was a clear reduction in free Sok-RNA (Figure 5A). The reduction in Sok-RNA over time was quantiﬁed and the binding-rate constant was 6.9 · 103 M 1 s 1 (37 C) (Figure 5B). There- fore, PNA::Sok-RNA interactions occur less quickly than hok mRNA::Sok-RNA interactions in vitro. The PAGE analysis did not reveal distinct PNA::Sok-RNA complexes, possibly due to the positively charged peptide attached to PNA and its effects on complex stability and migration during electrophoresis. These in vitro results suggest that efﬁcient Figure 4. Effect of anti Sok PNA treatment on hok mRNA processing. (A) hok mRNA isoforms. The 441 and 398 nt long RNAs are initial hok mRNAs that are processed into the mature, truncated isoform of 361 bp hok mRNA (15,19). In the presence of an excess of active Sok-RNA, the truncated version of the hok mRNA does not accumulate due to rapid Sok-RNA binding followed by rapid RNase III processing of the hok mRNA::Sok-RNA duplex. The long isoforms of hok mRNA bind Sok-RNA much slower and therefore accumulate even in the presence of Sok-RNA (26). The long versions of hok mRNA cannot bind to ribosomes and be translated and are not harmful to the cell. (B) Northern analysis of hok mRNAs following exposure to anti Sok PNA or rifampicin. The initial transcripts and 30 end truncated, mature and active isoforms of hok mRNA are indicated. Figure 5. PNA::Sok-RNA complex formation in vitro and binding kinetics. (A) 32P-Radiolabeled Sok-RNA incubated with a 15-fold molar excess of unlabelled anti Sok PNA (500 nM) or hok mRNA (30 nM), fractionated in a polyacrylamide/urea/TBE gel and analysed by autoradiography and densito- metry. (B) Plots of the fractions of uncomplexed Sok-RNA as a function of time of incubation, with the binding-rate constants indicated.