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.
The potential of the RNA phage MS2 to accommodate extra amino acids in its major coat protein has been examined. Accordingly, a pentapeptide was encoded in the genome as an N-terminal extension. In the MS2 crystal structure, this part of the coat protein forms a loop that extends from the outer surface of the icosahedral virion. At the RNA level, the insert forms a large loop at the top of an existing hairpin. This study shows that it is possible to maintain inserts in the coat protein of live phages. However, not all inserts were genetically stable. Some suffer deletions, while others underwent adaptation by base substitutions. Whether or not an insert is stable appears to be determined by the choice of the nucleic acid sequence used to encode the extra peptide. This effect was not caused by differential translation, because coat-protein synthesis was equal in wild-type and mutants. We conclude that the stability of the insert depends on the structure of the large RNA hairpin loop, as demonstrated by the fact that a single substitution can convert an unstable loop into a stable one.
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.
Hybrids between different species or genera of the single-stranded RNA coliphages have not been found in nature. Here, it has been shown that viable hybrids between different phage species can easily be generated in the laboratory by in vivo recombination. cDNA of species I phage MS2 located on a plasmid and lacking part of its 5h untranslated leader (5h UTR) was complemented with another plasmid carrying the 5h half of the genome of fr, a species I phage, or of KU1, a species II representative with low sequence similarity. When the two plasmids were present in the same cell there was spontaneous production of hybrid phages. Interestingly, these hybrids did not arise by a double or single crossover that would replace the missing MS2 sequences with those of fr or KU1. Rather, hybrids arose by attaching the complete 5h UTR of fr or KU1 to the 5h terminus of the defective MS2 phage. Several elements of the 5h UTR then occurred twice, one from KU1 (or fr) and the other from MS2. These redundant elements are in most cases deleted upon evolution of the hybrids. As a result, the 5h UTR of KU1 (or fr) then replaced that of MS2. It was earlier shown that this 5h UTR could assume two alternating structures that facilitated transient translation of the proximal maturation gene. Apparently, this timer function of the 5h UTR was exchangeable and could function independently of the rest of the genome. When hybrids were competed against wildtype, they were quickly outgrown, probably explaining their absence from natural isolates.
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