Qβ Replicase Discriminates between Legitimate and Illegitimate Templates by Having Different Mechanisms of Initiation

Abstract

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),...

Structure of the Qβ replicase, an RNA-dependent RNA polymerase consisting of viral and host proteins

“…1C, lanes 4 and 8), when only a 5-nt-long product strand might have been synthesized on the 3′-terminal template sequence, 5 0 -…UAGCCC-3 0 (3). The fact that such a short product remained tightly bound during the long electrophoresis procedure suggests that each of the RNA-bound bands represents the closed conformation of the replicative complex (17). When all four NTPs were present providing for the synthesis of full length product, the label was separated into a double-stranded product and high molecular weight complexes, which tend to be larger in the case of the dimer (Fig.…”

“…In our case, the basic catalytic machinery is well separated from the host proteins, which seem to provide a stabilizing scaffold for the β-subunit and perhaps provide interaction sites for the template both before and after initiation of replication. Qβ replicase is the most efficient in vitro system for nucleic acid amplification, far more efficient than PCR or isothermal amplification systems (17). However, the utilization of Qβ replicase for amplification of desired sequences has yet to be realized because of its puzzling and extraordinary template specificity requiring neither promoters nor primers (7,8).…”

Ribosomal protein S1 functions as a termination factor in RNA synthesis by Qβ phage replicase

“…During initiation on a legitimate template, Qb replicase undergoes GTP-triggered transition into a closed conformation, from which neither the template nor the product strand can dissociate until the full-sized product is synthesized 35 . Being advantageous for elongation of the initiated strands, the closed conformation must become an obstacle for the continuation of replication after elongation.…”

“…The used variant of RQ135 RNA was prepared by transcription of SmaI-digested plasmid pT7RQ135 -1 (-) 42 modified by inserting CGAUCC between positions 52 and 53 of the original RQ135 -1 (-) sequence; the template properties of this variant were indistinguishable 35 from those of the authentic RQ135 RNA 22 . Qb( þ ) RNA was isolated from purified wild-type phage particles lysed with SDS, followed by centrifugation through a 15% sucrose cushion and phenol extraction 25 .…”

“…Unless otherwise indicated, all reactions were run in 10 ml of the reaction buffer (100 mM HEPES-NaOH pH 7.5, 10 mM MgCl 2 , 1 mM EDTA, 0.1% Triton X-100) at 30°C, in the presence of 1 mM each of NTPs. The reactions were stopped by the addition of 10 ml of 20 mM EDTA, treated or not with ribonuclease T1 as indicated, extracted with 20 ml of phenol/chloroform (1:1, v/v), and 10 ml of the aqueous phase was subjected to nondenaturing electrophoresis through a 8% polyacrylamide gel 35 in case of RQ RNA or 1% agarose gel in case of Qb RNA. The 32 P-labeled RNA bands on dried gels were revealed using a Cyclone phosphor storage system (Packard Instrument) and either presented here as original images, or quantified by measuring the band intensities 43 .…”