Extensive nonhomologous recombinations occur between the 5' and 3' fragments of a replicable RNA in a cell-free system composed of pure Qbeta phage replicase and ribonucleoside triphosphates, providing direct evidence for the ability of RNAs to recombine without DNA intermediates and in the absence of host cell proteins. The recombination events are revealed by the molecular colony technique that allows single RNA molecules to be cloned in vitro. The observed nonhomologous recombinations are entirely dependent on the 3' hydroxyl group of the 5' fragment, and are due to a splicing-like reaction in which RNA secondary structure guides the attack of this 3' hydroxyl on phosphoester bonds within the 3' fragment.
The ability of RNAs to spontaneously rearrange their sequences under physiological conditions is demonstrated using the molecular colony technique, which allows single RNA molecules to be detected provided that they are amplifiable by the replicase of bacteriophage QL L. The rearrangements are Mg 2+ -dependent, sequence-non-specific, and occur both in trans and in cis at a rate of 10 39 h 31 per site. The results suggest that the mechanism of spontaneous RNA rearrangements differs from the transesterification reactions earlier observed in the presence of QL L replicase, and have a number of biologically important implications.z 1999 Federation of European Biochemical Societies.
S1 is the largest ribosomal protein, and is vitally important for the cell. S1 is also a subunit of Qb replicase, the RNA-directed RNA polymerase of bacteriophage Qb. In both protein and RNA syntheses, S1 is commonly believed to bind to a template RNA at the initiation step, and not to be involved in later events. Here, we show that in Qb replicase-mediated RNA synthesis, S1 functions at the termination step by promoting release of the product strand in a single-stranded form. This function is fulfilled by the N-terminal fragment comprising the first two S1 domains. The results suggest that S1 might also have a role other than mRNA binding in the ribosome.
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