FIG. 4.

Initiation of genomic plus-strand RNAs directed by minus-strand endscripts. (A) Comparison of the 3′ sequences of BMV and CMV minus-strand RNAs. The nontemplated guanylate added to each template is shown in bold type. The initiation cytidylate is denoted by an arrow. (B) Predicted secondary structures of the 3′ ends of BMV RNA1, RNA2, and RNA3, i.e., B1(−)58G, B2(−)46G, and B3(−)51G, respectively. The structure predictions were generated by the MFOLD program (10). (C) Initiation of genomic plus-strand RNAs from minus-strand endscripts. RdRp reaction products were separated by denaturing PAGE (12% polyacrylamide) and visualized by autoradiography. The amounts of RNA synthesized from various templates relative to B2(−)46G (% Syn) are shown at the bottom of the autoradiogram. The results presented are an average from three independent trials. The sizes of the RNA products are indicated on the side of the autoradiogram. The symbol φ represents the products of a control reaction with no added template. Endscripts that are initiation competent are indicated by +, while initiation-incompetent endscripts are indicated by −. C2(−)G and C3(−)G are endscripts of CMV RNA2 and CMV RNA3, respectively. (D) Synthesis of a 200-nt genomic plus-strand RNA. The endscript with a guanylate replacing the +1 cytidylate is indicated by +1c/g. −G denotes a 200-nt endscript without the designed nontemplated guanylate; +G denotes a 200-nt endscript with a guanylate at the 3′ end of the RNA. RNA synthesis from B2(−)200+G, B2(−)200Δ-1, and B2(−)200+1C/G was 100, 27, and 0% respectively. The results presented are an average from three independent trials. M denotes a reaction designed to produce a molecular mass marker of 203 nt.