Translational control by delayed RNA folding: identification of the kinetic trap.

The maturation or A-protein gene of single-stranded RNA phage MS2 is preceded by a 130-nt long untranslated leader. When MS2 RNA folding is at equilibrium, the gene is untranslatable because the leader adopts a well-defined cloverleaf structure in which the Shine-Dalgarno (SD) sequence of the maturation gene is taken up in long-distance base pairing with an upstream complementary sequence (UCS). Synthesis of the A-protein takes place transiently while the RNA is synthesized from the minus strand. This requires that formation of the inhibitory cloverleaf is slow. In vitro, the folding delay was on the order of minutes. Here, we present evidence that this postponed folding is caused by the formation of a metastable intermediate. This intermediate is a small local hairpin that contains the UCS in its loop, thereby preventing or slowing down its pairing with the SD sequence. Mutants in which the small hairpin could not be formed made no detectable amounts of A-protein and were barely viable. Apparently, here the cloverleaf formed quicker than ribosomes could bind. On the other hand, mutants in which the small intermediary hairpin was stabilized produced more A-protein than wild type and were viable. One hardly growing mutant that could not form the metastable hairpin and did not make detectable amounts of A-protein was evolved. The emerging pseudo-revertant had selected two second site repressor mutations that allowed reconstruction of a variant of the metastable intermediate. The pseudo-revertant had also regained the capacity to produce the A-protein.

In vivo oligo(A) insertions in phage MS2: role of Escherichia coli poly(A) polymerase.

Previously we introduced an RNase III site into the genome of RNA phage MS2 by extending a hairpin with a perfect 18 bp long stem. One way in which the phage escaped from being killed by RNase III cleavage was to incorporate uncoded A residues on either side of the stem. This oligo(A) stretch interrupts the perfect stem that forms the RNase III site and thus confers resistance. In this paper we have analyzed the origin of these uncoded adenosines. The data strongly suggest that they are added by the host enzyme poly(A) polymerase. Apparently the 3'-OH created by RNase III cleavage becomes a substrate for poly(A) polymerase. Subsequently, MS2 replicase makes one contiguous copy from the two parts of the genome RNA. The evolutionary conversion from RNase III sensitivity to resistance provides a large spectrum of solutions that could be an important tool to understand what essentially constitutes an RNase III site in vivo.

The in vitro more efficiently repaired cisplatin adduct cis-Pt.GG is in vivo a more mutagenic lesion than the relative slowly repaired cis-Pt.GCG adduct.

The toxic effect and the mutagenicity of two differentially repaired site-specific cis-diamminedichloroplatinum(II) (cis-DDP) lesions were investigated. Detailed analysis of the UvrABC-dependent repair of the two lesions in vitro showed a more efficient repair of the cis-Pt.GG adduct compared to that of the cis-Pt.GCG adduct (Visse et al., 1994). Furthermore, previously, a dependency of cis-DDP mutagenesis on UvrA and UvrB, but not on UvrC was found (Brouwer et al., 1988). To possibly relate survival and mutagenesis to repair, plasmids containing the same site-specific cis-DDP lesions as those that were used in the detailed repair studies were transformed into Escherichia coli. The results indicate that both lesions are very efficiently bypassed in vivo. Mutation analysis was performed using a denaturing gradient gel electrophoresis technique, which allows identification of mutations without previous selection. Although the cis-Pt.GG adduct is in vitro more efficiently repaired than the cis-Pt.GCG adduct, it appeared to be more mutagenic. We present a model in which this result is related to the previously observed dependency of the mutagenicity of cis-DDP lesions on the Uvr A and B proteins.