Functional circularity of legitimate Qbeta replicase templates

Abstract

Qbeta replicase (RNA-directed RNA polymerase of bacteriophage Qbeta) exponentially amplifies certain RNAs in vitro. Previous studies have shown that Qbeta replicase can initiate and elongate on a variety of RNAs; however, only a minute fraction of them are recognized as 'legitimate' templates. Guanosine 5'-triphosphate (GTP)-dependent initiation on a legitimate template generates a stable replicative complex capable of elongation in the presence of aurintricarboxylic acid, a powerful inhibitor of RNA-protein interactions. On the contrary, initiation on an illegitimate template is GTP independent and does not result in the aurintricarboxylic-acid-resistant replicative complex. This article demonstrates that the 3' and 5' termini of a legitimate template cooperate during and after the initiation step. Breach of the cooperation by dividing the template into fragments or by introducing point mutations at the 5' terminus reduces the rate and the yield of initiation, increases the GTP requirement, decreases the overall rate of template copying, and destabilizes the postinitiation replicative complex. These results revive the old idea of a functional circularity of legitimate Qbeta replicase templates and complement the increasing body of evidence that functional circularity may be a common property of RNA templates directing the synthesis of either RNA or protein molecules.

Fig. 1

Putative secondary structures of RQ135 RNA and of a hybrid molecule formed by its 3′ and 5′ fragments. (a) A model accounting for the results of ribonuclease probing. (b) A model generated by program RNA mfold 3.2 [http://frontend.bioinfo.rpi.edu/applications/mfold/cgi-bin/rna-form1.cgi] based on a minimum energy algorithm., (c) A model of interacting 3′ fragment (black symbols) and 5′ fragment (green symbols) patterned upon model (a). Bases not present in the wild-type RQ135 RNA are shown with lowercase letters. Blue symbols indicate the 3′-terminal segment responding to ribonucleases as a double helix. Red symbols indicate the 5′-terminal bases subjected to site-directed mutagenesis.

Fig. 2

Initial kinetics of RNA synthesis on the unbroken RQ135 RNA, with the wild-type (GGG, blue line with filled symbols) or mutant (GAA, red line with filled symbols) 5′ terminus, and on its 3′ fragment, alone (black line with filled symbols) or hybridized with the 5′ fragment possessing the wild-type (GGG, blue line with unfilled symbols) or mutant (GAA, red line with unfilled symbols) 5′ terminus. Each line represents the least-squares fit for linear equation (y = mx + b). The broken line was obtained from the blue line with unfilled symbols by multiplying it with 42/34, a ratio of the number of G residues (directing the incorporation of [³²P]CMP) in the RQ135 RNA and its 3′ fragment, respectively. The ordinate displays amounts of the full-sized product strands [expressed in digital light units (DLU)] in PAGE patterns shown in Supplementary Fig. S3. In this experiment, 1 pmol of incorporated [³²P]CMP corresponded to approximately 20 × 10⁶ DLU.

Fig. 3

Effects of 5′-terminal point mutations on GTP dependence of RNA synthesis on (a) RQ135 RNA; (b) its 3′ fragment, alone or hybridized with the 5′ fragment; and (c) the 5′ fragment. The schemes on the left show location of the mutations (indicated with lowercase letters). Synthesis in (a) and (b) was performed in two steps: a 10-min incubation in the reaction mixture containing GTP (initiation) followed by the addition of a mixture of the missing rNTPs and ATA (to 1 mM final concentration) and further incubation for 10 min (elongation). Indicated are the final GTP concentrations at the elongation step; during initiation, the concentration of all reaction components including GTP was 25% higher. Synthesis in (c) was performed in one step (see Materials and Methods), with the GTP concentration being varied as indicated.

Fig. 4

Time course of the initiation of RNA synthesis on RQ135 RNA with the wild-type (GGG, blue lines) or mutant (GAA, red lines) 5′ terminus at two different GTP concentrations. Synthesis was performed in two steps (see legend to Fig. 3), with the initiation time being varied and the GTP concentration fixed as indicated.

Fig. 5

Time course of decay in the presence of ATA of replicative complexes formed on the unbroken RQ135 RNA (a–c) or on its 3′ fragment (d). Complexes were generated by a 10-min initiation in the presence of GTP. Decay was initiated by the addition of ATA. After the indicated time intervals, a mixture of the missing rNTPs was added, and incubation was continued for 10 min (elongation). The final concentration of each rNTP and ATA was 1 mM. The amounts of full-sized RNAs produced by the residual elongation-competent postinitiation complexes are plotted against time on a linear (a) or exponential (b–d) scale. (a and b) The 5′ terminus of RQ135 RNA was of the wild type (GGG) or carried the indicated mutations. (c) The 3′ terminus of RQ135 RNA carried the indicated mutations, with the 5′ terminus either being of wild type or carrying complementary mutations, as indicated in legends of matching colors. (d) Decay of the complexes formed by the 3′ fragment alone (black lines), whose 5′ terminus was wild type (GGC, filled symbols) or mutant (GGG, unfilled symbols), or by the 3′ fragment hybridized with the 5′ fragment (colored lines), which was of the wild type or carried 5′-terminal mutations, as indicated in legends of matching colors. Gray lines in (c) and (d) are replicas of the lines from (b). Each line represents the least-squares fit through points by using the power equation (y = ce^bx).

Fig. 6

Products of RNA synthesis on RQ135 RNA, whose 5′ and 3′ trinucleotides (separated by a slash) were of the wild type or contained mutations indicated with lowercase letters. Synthesis was performed in two steps (see legend to Fig. 3). Initiation was performed in the presence of GTP (a) or GTP and ATP (b), whose final concentration was 1 mM each. Arrows indicate the bands corresponding to a ds and ss product.