ИСТИНА

Bacteriophage Qβ is a positive-strand RNA virus which infects E. coli. Its propagation depends on recruitment of host proteins that normally take part in protein synthesis. Three of them switch to RNA replication by forming viral RNA-depended RNA polymerase (Qβ replicase) together with viral-encoded β-subunit after infection. This enzyme is remarkable because it can exponentially amplify RNA in vitro at an unprecedented rate. Synthesis of GGG, complementary to the 3'-terminal CCC of the template, drives the replicase into a closed conformation, from which the growing RNA strand cannot dissociate. Termination (release of the completed RNA strand) is promoted by the ribosomal protein S1, one of the Qβ replicase subunits, as well as by its N-terminal fragment OB(1-2). We discovered that with GTP as the only substrate, Qβ replicase produces long polyG strands, which on denaturing gel electrophoresis produce a ladder with at least three clusters of bolder bands of about 15, 25 and 35 nt. Varying the GTP concentration or incubation time changes the distribution of material among the clusters, but the positions of the clusters in the gel remain preserved. This synthesis is template-directed, it only occurs in the closed replicase conformation, and is prevented by incorporation of the next template-encoded nucleotide; the latter indicates that it results from transcript slippage. This is the first time that slippage is demonstrated for the replicase of a positive strand RNA phage, and the first time ever that transcript slippage is found to generate products whose amount periodically changes with their size. The most intriguing observation is that protein S1 and its fragment OB(1-2) promote release of the G15 product suggesting that they recognize it as a termination signal. In view of the known propensity of G-rich sequences to form quadruplexes, this indicates that a G quadruplex-like structure may be formed by the replicative complex at the termination step.