Autonomous role of 3'-terminal CCCA in directing transcription of RNAs by Qbeta replicase

Abstract

We have studied transcription in vitro by Qbeta replicase to deduce the minimal features needed for efficient end-to-end copying of an RNA template. Our studies have used templates ca. 30 nucleotides long that are expected to be free of secondary structure, permitting unambiguous analysis of the role of template sequence in directing transcription. A 3'-terminal CCCA (3'-CCCA) directs transcriptional initiation to opposite the underlined C; the amount of transcription is comparable between RNAs possessing upstream (CCA)(n) tracts, A-rich sequences, or a highly folded domain and is also comparable in single-round transcription assays to transcription of two amplifiable RNAs. Predominant initiation occurs within the 3'-CCCA initiation box when a wide variety of sequences is present immediately upstream, but CCA or a closely similar sequence in that position results in significant internal initiation. Removal of the 3'-A from the 3'-CCCA results in 5- to 10-fold-lower transcription, emphasizing the importance of the nontemplated addition of 3'-A by Qbeta replicase during termination. In considering whether 3'-CCCA could provide sufficient specificity for viral transcription, and consequently amplification, in vivo, we note that tRNA(His) is the only stable Escherichia coli RNA with 3'-CCCA. In vitro-generated transcripts corresponding to tRNA(His) served as poor templates for Qbeta replicase; this was shown to be due to the inaccessibility of the partially base-paired CCCA. These studies demonstrate that 3'-CCCA plays a major role in the control of transcription by Qbeta replicase and that the abundant RNAs present in the host cell should not be efficient templates.

FIG. 1

Inactivation of the 3′-penultimate CCA initiation box does not increase initiation from the 3′-CCA initiation box. RNA variants derived from CCA9 RNA, bearing the mutations of the C residues in initiation box #8 shown in panel A, were tested as templates with Qβ replicase. RNAs were incubated in the presence of Qβ replicase and [α-³²P]CTP for 10 min at 37°C as described in Materials and Methods, except that MgCl₂ was present at 21 mM. Products made from templates identified by their initiation box #8 sequence were separated by 12.5% denaturing PAGE (shown in panel B). The numbers at the left identify the CCA repeat (initiation box numbers) from which product strands originate, #9 representing the 3′-most CCA. Each CCA initiation box produces three products, marked a, b, and c at the left, whose origins are explained in the text.

FIG. 2

A CCCA 3′-initiation box directs strong 3′-terminal initiation from a wide range of adjacent sequences. (A) RNAs in which the 3′-CCACCA of CCA9 RNA was changed to 3′-NNNCCCA were incubated in the presence of Qβ replicase and [α-³²P]CTP for 10 min at 37°C as described in Materials and Methods, with MgCl₂ present at 10 mM. As indicated, many of the 3′-heptanucleotide sequences were derived from the 3′ termini of RNAs exponentially amplifiable by Qβ replicase: Qβ genome(+), WSI (26); Qβ genome(−) (34); MDV-1 (24); DN3 (40); RQ120 (21); 50#1, 50#2, and 77#1 (7); MNV-1 (2); SV7 (GenBank accession no. L07339); and SV11 (GenBank accession no. L07337). (B) Analysis of transcription products labeled with [α-³²P]CTP and separated by 12.5% denaturing PAGE, with templates identified by the sequence of their modified initiation box #8. The relative levels of transcription (with reference to box #8 of CCA9) originating from box #9 of each RNA is given at the foot of each lane (average of three experiments; typical standard deviation = 10 to 20%). Below the panel is shown the proportion (% of total) of transcripts >12 nt in length originating from box #9 (box #8 for CCA9). ✽, Quantitation of transcription from box #8 in the case of CCA9 RNA.

FIG. 3

C_3-5A sequences provide optimal 3′ initiation sites. The RNAs shown in panel A were analyzed as transcriptional templates for Qβ replicase (panel B) as described in Fig. 2. The relative levels of transcription (% of total, with reference to box #8 of CCA9) originating from boxes #8 and #9 of each RNA are given below each lane (average of three experiments; typical standard deviation = 10 to 20%); note that for lanes 14 and 15, the numbers refer to initiation from the 3′ and 3′-penultimate boxes, respectively. Below these figures are shown the proportions of transcripts >12 nt in length originating from box #9 (as well as box #8 for CCA9) as a percentage.

FIG. 4

Upstream CCA boxes contribute little to initiation from a 3′-CCCA sequence. (A) Derivatives of AAACCCA RNA in which CCA boxes #1 to #7 have been replaced progressively with A-rich sequences (underlined). (B) Analysis of the same RNAs as transcriptional templates for Qβ replicase, performed as described in Fig. 2. The dots placed to the left of lanes indicate absent signals from the mutated initiation boxes. The relative level of transcription (percentage of total, with reference to box #8 of CCA9) originating from box #9 of each RNA is given below each lane (average of three experiments; typical standard deviation = 10 to 20%), as is the proportion (percentage of total) of transcripts >12 nt in length originating from box #9.

FIG. 5

Role of the 3′-terminal A residue. Two families of RNAs are analyzed as transcriptional templates for Qβ replicase as described in Fig. 2. In lanes 2 to 4, derivatives of AAA72 RNA with the indicated 3′ ends are analyzed: the AAA72 RNA family has the sequence GGA₅UA₆UA₅UA₆ upstream of the indicated C-rich initiation box. In lanes 6 to 9, derivatives of GGA(CCA)₇X RNA, with the indicated 3′ ends representing X, are analyzed. The major end-to-end transcription product is marked N at the right side of the figure, with the upper band evident in lanes 8 and 9 marked N+1. The entire cluster of bands originating from the initiation boxes of the RNAs in lanes 6 to 9 is bracketed and labeled “C.” The relative percent transcription yields are given below each lane, separately for C, N, and N+1 in panel B. The yields of products in lanes 3 and 6 were 170 and 125%, respectively, relative to transcription from box #8 of CCA9 RNA. The RNAs in lanes 6 to 9 were previously analyzed (38) at 21 mM MgCl₂, which favors internal over 3′-end initiation.

FIG. 6

E. coli tRNA^His is a poor template for Qβ replicase because its 3′ terminus is unavailable. (A) Sequence of tRNA^His (lacking posttranscriptional modifications). The arrow marks the additional 5′-nucleotide (G) that is unique to tRNA^His and that is absent from the RNA template used in lane 3 of panel B. The bracket encompasses the 5′-GGU missing from the template tested in lane 4. The additional 3′ sequence present on the RNA tested in lane 5 is shown in italics. (B) Transcription products generated from the indicated templates after incubation with Qβ replicase as described in Fig. 2, except that analysis is by 10% denaturing PAGE. The relative molar transcription levels (Rel. tr. [percent], with reference to box #8 of CCA9, 100*) originating from each RNA is given at the foot of each lane (average of three experiments; typical standard deviation = 10 to 20%).

FIG. 7

Transcription from a 3′-UCCA terminus. Transcription from GGA(CCA)₇CCUUCCA and CCA9 RNAs by Qβ replicase was compared as described in Fig. 2. CCA9 RNA (30 nt) is the shorter of the two RNAs by 1 nt.