Spontaneous rearrangements in RNA sequences.
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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 Qbeta. The rearrangements are Mg2+-dependent, sequence-non-specific, and occur both in trans and in cis at a rate of 10(-9) h(-1) per site. The results suggest that the mechanism of spontaneous RNA rearrangements differs from the transesterification reactions earlier observed in the presence of Qbeta replicase, and have a number of biologically important implications. (+info)
The puzzle of RNA recombination.
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For more than three decades, RNA recombination remained a puzzle and has only begun to be solved in the last few years. The available data provide evidence for a variety of RNA recombination mechanisms. Non-homologous recombination seems to be the most common for RNA. Recent experiments in both the in vitro and the in vivo systems indicate that this type of recombination may result from various transesterification reactions which are either performed by RNA molecules themselves or are promoted by some proteins. The high frequency of homologous recombination manifested by some RNA viruses can be easier explained by a replicative template switch. (+info)
Minimal requirements for template recognition by bacteriophage Qbeta replicase: approach to general RNA-dependent RNA synthesis.
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Any oligo- or polynucleotide able to offer a C-C-C-sequence at the 3'-terminus and a second C-C-C-sequence in a defined steric position to Qbeta replicase is an efficient template. Corresponding chemical modifications convert non-template RNAs to template RNAs. (+info)
Nucleotide sequence of microvariant RNA: another small replicating molecule.
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Microvariant RNA, a small self-replicating molecule (114 nucleotides long), has been isolated from Qbeta replicase reactions incubated in the absence of exogenous template. Its complete nucleotide sequence has been determined. Comparison with MDV-1 RNA, a somewhat larger endogenous Qbeta replicase product (220 nucleotides long) that had previously been characterized, revealed no significant sequence similarity. Since Qbeta replicase can mediate the synthesis of both of these disparate RNA molecules, primary sequence cannot be the sole determining factor in the processes of enzyme recognition and replication. This implies that the key is to be found in the secondary or tertiary structures. The availability of two different replicating molecules of defined sequence should aid in identifying these critical structural features. (+info)
Reconstitution of Qbeta RNA replicase from a covalently bonded elongation factor Tu-Ts complex.
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Escherichia coli phage Qbeta RNA replicase, an RNA-dependent RNA polymerase (RNA-dependent RNA nucleotidyltransferase), is a tetramer composed of one phage-coded polypeptide and three host-supplied polypeptides which are known to function in the biosynthesis of proteins in the uninfected host. Two of these polypeptides, protein synthesis elongation factors EF-Tu and EF-Ts, can be covalently crosslinked with dimethyl suberimidate to form a complex which lacks the ability to catalyze the known host functions catalyzed by the individual elongation factors. Using a previously developed reconstitution system we have examined the effects of crosslinking the EF-Tu-Ts complex on reconstituted replicase activity. Renaturation is significantly more efficient when exogenously added native EF-Tu-Ts is crosslinked than when it is not. Crosslinked EF-Tu-Ts can be purified from a crude crosslinked postribosomal supernatant by its ability to replace EF-Tu and EF-Ts in the renaturation of denatured Qbeta replicase. A sample of Qbeta replicase with crosslinked EF-Tu-Ts replacing the individual elongation factors was prepared. Although it lacked EF-Tu and EF-Ts activities, it could initiate transcription of both poly(C) and Qbeta RNA normally and had approximately the same specific activity as control enzyme. Denatured Qbeta replicase formed with crosslinked EF-Tu-Ts was found to renature much more rapidly than untreated enzyme and, in contrast to normal replicase, its renaturation was not inhibited by GDP. The results demonstrate that EF-Tu and EF-Ts function as complex in Qbeta replicase and do not perform their known protein biosynthetic function in the RNA synthetic reaction. (+info)
Internal and 3' RNA initiation by Qbeta replicase directed by CCA boxes.
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RNA initiation by Qbeta replicase directed by the short-sequence CCA at the 3'-end of all RNAs amplified by this enzyme has been studied. Most CCA repeats in an RNA consisting of 12 CCAs serve as independent sites of de novo RNA initiation, with initiation occurring opposite the 3'-C residue of each CCA. Qbeta replicase is thus capable of internal initiation remote from the 3'-end, although predominant initiation occurs close to the 3'-end. The precise 3'-terminal sequence in (CCA)(n)-containing RNAs influences the number and position of active initiation sites near the 3'-terminus. C residues are required at the initiation site, whereas the position of purines (especially A residues) influences the selection of initiation sites. The template activity of (CCA)(n) RNAs is positively correlated with the number of CCA repeats. Three CCA repeats added to the 3'-end of a nontemplate 83-nt RNA are sufficient to activate transcription by Qbeta replicase. These experiments show that CCA boxes can act as strong initiation sites in the absence of specific cis-acting signals derived from Qbeta RNA, although the efficiency of initiation is modulated by surrounding sequence. (+info)
Initiation specificity of the poly(cytidylic acid)-dependent Qbeta replicase activity.
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The initiation specificity of RNA synthesis catalysed by the poly(C)-dependent Qbeta replicase activity was investigated with various synthetic ribopolymers as template. It was found that the initiation efficiency of a series of oligo(C) with various chain lengths is proportional to the template size. Synthetic riboheteropolymers containing cytidylic acid were accepted as templates only if they contained at their 3' end a cytidylic acid sequence of more than 5 nucleotides. Such an oligocytidylate sequence served also as an initiator sequence for copying the non-cytidylic-acid-containing part of the heterotemplate. RNA synthesis always began with the incorporation of GTP, even if the 3'-terminating nucleotide of the template was not cytidylic acid. (+info)
Phage Qbeta replicase: cell-free synthesis of the phage-specific subunit and its assembly with host subunits to form active enzyme.
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Cell-free translation of Qbeta RNA and subsequent partial purification of the enzyme resulted in replicase activity. From 0.5 to 1.5% of all R chains synthesised were found in the 7-S replicase complex. The presence in the 7-S complex of the host subunits of authentic replicase, i (= S1) and EF-Ts, was shown by the effect of antisera directed against ribosomal protein S1 and EF-Ts, respectively. Furthermore, the presence of EF-Ts was demonstrated by thermal denaturation of in vitro replicase made by a cell extract from an Escherichia coli mutant with a thermolabile EF-Ts. In vitro replicase did not assemble spontaneously during protein synthesis but was formed upon subsequent purification. Assembly could be induced by ammonium sulphate precipitation (60% saturation) alone. It is concluded that the functional phage-coded subunit synthesised in vitro recognises i and the EF-Tu - EF-Ts complex among a mixture of host proteins. (+info)