Synergism in replication and translation of messenger RNA in a cell-free system.

Abstract

Combination of the Qf8 replicase reaction with the Escherichia coli cell-free translation system markedly enhances replication of a recombinant RQ-DHFR RNA consisting of the dihydrofolate reductase (DHFR) mRNA sequence inserted into RQ135-1 RNA, an efficient naturally occurring Q13 replicase template. The enhancement is associated with a replication asymmetry previously described for the replication of Qj3 phage RNA in vivo; the sense (+)-strands are produced in large excess over the antisense (-)-strands. This, in turn, results in increased synthesis of the functionally active DHFR. These effects are not observed when DHFR mRNAs or RQ135-1 RNAs are used as templates, if the translation system is not complete, or if it is inhibited by puromycin. The coupled replication-translation of nonviral mRNA recombinants can serve as a useful model for studying the fundamental aspects of virus amplification and can be implemented for large-scale protein synthesis in vitro. We examined the effects ofintracellular components on the capability of an RQ-mRNA recombinant to replicate in vitro and found that significant stimulation of RNA synthesis is only observed in the presence of a complete functioning translation system. The stimulation is associated with elevated production of the sense (+)-strands over the antisense (-)-strands, which in turn provides more messengers for translation. Thus, coupling of the replication and translation reactions results in their synergistic action. MATERIALS AND METHODSTemplate RNAs. RQ-DHFR RNA recombinants were prepared at the DNA level by inserting the bacterial dihydrofolate reductase (DHFR) mRNA sequence into the sequence of RQ135-1(-) RNA (18). The sense (+)-and antisense (-)-strands of RQ-DHFR RNA were obtained by runoff transcription (19) of plasmids pT7RQ135-1(-)DHFR(+) or pT7RQ135-1(-)DHFR(-), respectively, digested with endonuclease Sma I. The plasmids were prepared as follows. A DHFR gene-containing Sma I/Hae II fragment was excised from plasmid pSP65DHFR generously provided by N. V. Murzina (this institute) and treated with T4 DNA polymerase to produce blunt ends prior to ligation into site Xho I of a mutant plasmid pT7RQ135-1(-), which was blunt-ended by incubation with Klenow fragment. The original pT7RQ135-1(-) was prepared by A. V. Munishkin (this institute) by insertion of a PCR-amplified T7 promoter/ RQ135S1(-) cDNA construct into plasmid pUC18 between polylinker sites HindIII and Sma I. To generate a unique Xho I site within pT7RQ135-1(-), the T --C and A --T substitutions at positions 53 and 54 of the RQ135-1(-) sequence, respectively, were introduced by oligonucleotide mutagenesis.Control DHFR mRNA containing a 36-nt-long 5'-terminal and a 165-nt-long 3'-terminal untranslated region originating from the E. coli chromosome (20)

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

“…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 .…”

Expression and stability of recombinant RQ‐mRNAs in cell‐free translation systems

“…Plasmids pT7RQ135_,(-)DHFR(+) (coding the recombinant RQ-DHFR mRNA) and pSP65DHFR,,, (used for synthesis of the control DHFR mRNA with SP6 RNA polymerase) were the same as in [9]. Plasmid pSPT19CAT (generously provided by Dr. Yarchuk of this Institute) was prepared by ligation, at site SalI plasmid pSPT19 (Pharmacia), of the CAT gene-carrying SalI cartridge excised from plasmid pCM-1 (Pharmacia).…”

“…RQ mRNA recombinants were constructed at the DNA level by inserting the corresponding genes into the XhoI site of mutant RQ135_, cDNA and prepared by runoff plasmid transcription with T7 RNA polymerase as described [9]. Plasmids pT7RQ135_,(-)DHFR(+) (coding the recombinant RQ-DHFR mRNA) and pSP65DHFR,,, (used for synthesis of the control DHFR mRNA with SP6 RNA polymerase) were the same as in [9].…”

“…Otherwise, replication is limited to one round resulting in a dead duplex formed by the annealed (+) and (-) strands. The translating ribosomes prevent duplex formation by sequestering the (+) strands and induce replication asymmetry that also characterizes the replication of QB RNA in vivo: the (+) strands are accumulated in large excess over the (-) strands, and this results in an increased synthesis of DHFR [9]. Thus, the behavior of the recombinant RQ-DHFR mRNA in the cell-free replicationtranslation system mimics the replication of QB phage in vivo, although no part of RQ-DHFR mRNA has originated from the phage genome.…”

“…In a previous study we have shown that RQ135 RNA can impart the ability of amplification by QJ3 replicase in vitro to DHFR mRNA inserted at one of its internal loops [9]. Replication of the RQ-DHFR mRNA recom-*Corresponding author.…”