An in vivo recombinant RNA capable of autocatalytic synthesis by Qβ replicase

Abstract

A variety of small RNAs ranging from tens to hundreds of nucleotides in length grow autocatalytically in a Q beta replicase (Q beta phage RNA-dependent RNA polymerase) reaction in the absence of added template, and similar RNAs are found in Q beta phage-infected Escherichia coli cells. Three such RNAs have been sequenced. One of them that is 221 nucleotides (nt) long ('MDV-1' RNA) has been found to be partially homologous to Q beta phage RNA 8, which might be considered as an indication of its origination from by-products of the Q beta RNA replication. To gain further insight into the origin and function of these RNAs, we have sequenced a new RNA, 120 nt long, isolated from the products of spontaneous synthesis by the nominally RNA-free Q beta replicase preparation. The minus strand of this RNA appeared to be a recombinant RNA, composed of the internal fragment of Q beta RNA (approximately 80 nt long) and the 33-nt-long 3'-terminal fragment of E. coli tRNA(1Asp). This seems to be the first strong indication of RNA recombination in bacterial cells. The various implications of this finding are discussed.

tRNA‐like structures

“…These structural features that deviate from canonical tRNA may explain why attempts to aminoacylate coliphage RNA have remained unsuccessful. Putative aminoacyl acceptor domain at the 3' end of RNA coliphages (Adhin et al, 1990 Qp virus, called RQ120 (Munishkin et al, 1988). The tRNAASp moiety of RQ120 has its 3' terminus covalently attached to the viral RNA like the tRNAASp moiety of some DI RNAs of Sindbis virus (see above).…”

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

“…The recombinant RQ-mRNAs used in this work contained E. coli DHFR and CAT mRNAs inserted between positions 53 and 54 of the (-) strand of RQ135_, RNA [2]. In addition to the 480 nt-and 657 nt-long sequences coding for DHFR and CAT, the inserts contained, respectively, the 15 nt-and 37 nt-long 5' terminal, and the 22 nt-and 93 nt-long 3' terminal untranslated segments.…”

“…These RNAs possess highly developed secondary structures and, presumably, stable molecule 'stalks' formed by the base-paired 3' and 5' termini [ 1,2,15]. The latter feature can be important for RNA protection against 3'-exoribonucleases which are abundant in bacterial cells and play a major role in mRNA turnover in vivo [16].…”

“…by exposing the ribosome-binding site. (ii) Another possibility could be accounted for by the fact that the (-) strand of RQ135 RNA, in which the mRNAs have been inserted, contains three long segments complementary to the 23 S ribosomal RNA [2] that could provide for additional binding sites of the recombinant RQ-mRNAs on the ribosome.…”

“…Recently, we have shown that the 'spontaneous' synthesis is caused by airborne RQ RNA molecules [4,8] and that RQ RNAs do not necessarily descend from the Q/3 RNA sequence. In particular, RQ135(+) RNA, one of the most efficient Qj3 replicase templates, is mainly composed of sequences homologous to the 23 S ribosomal RNA [2].…”