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.
Q replicase (RNA-directed RNA polymerase of bacteriophage Q) exponentially amplifies certain RNAs (RQ RNAs) in vitro. Here we characterize template properties of the 5 and 3 fragments obtained by cleaving one of such RNAs at an internal site. We unexpectedly found that, besides the 3 fragment, Q replicase can copy the 5 fragment and a number of its variants, although they lack the initiator region of RQ RNA. This copying can occur as a 3-terminal elongation or through de novo initiation. In contradistinction to RQ RNA and its 3 fragment, initiation on these templates occurs without regard to the 3-terminal or internal oligo(C) clusters, is GTP-independent, and does not result in a stable replicative complex capable of elongation in the presence of aurintricarboxylic acid. The results suggest that, although Q replicase can initiate and elongate on a variety of RNAs, only some of them are recognized as legitimate templates. GTP-dependent initiation on a legitimate template drives the enzyme to a "closed" conformation that may be important for keeping the template and the complementary nascent strand unannealed, without which the exponential replication is impossible. Triggering the GTP-dependent conformational transition at the initiation step could serve as a discriminative feature of legitimate templates providing for the high template specificity of Q replicase.Q replicase, the RNA-directed RNA polymerase of bacteriophage Q, amplifies the 4217-nt 1 -long genomic Q RNA and a number of RQ RNAs, which are usually Յ250 nt in length. The natural source of RQ RNAs is Q phage itself or Q phageinfected Escherichia coli cells where these RNAs are formed by recombination from viral and/or cellular RNAs and propagated (1-3). Recently, many new RQ RNAs have been selected from random (4) or artificially designed (5) sequences, or produced by in vitro RNA recombination (6, 7). The amplification of these RNAs is exponential as long as the enzyme is in molar excess: the number of RNA molecules doubles in each round of replication, because both the original RNA and its complementary copy are replicase templates. Approximately 10 4 copies of a single genomic RNA molecule are produced in a Q phageinfected E. coli cell in less than 1 h (8). Amplification of small RQ RNAs is much faster: up to 10 10 copies are produced at room temperature within 10 min in a cell-free system comprised of purified Q replicase and all four NTPs (3), and this is the absolute record of the rate of nucleic acid amplification. The high amplification rate allows single RQ RNA molecules to rapidly produce detectable molecular colonies if they are amplified in a gel (9, 10).However, Q replicase does not amplify most RNAs, including any tested cellular RNAs or genomic RNAs of other viruses. Selection experiments indicated that only a few of the initial diversity of 10 12 unique sequences of 50 -77 nt in length are replicable (4), demonstrating a very high degree of template specificity of the enzyme. Now, almost 40 years since its discovery (11),...
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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