FIG. 3.

VPg-linked poly(U) products from wild-type and CRE-independent negative-strand RNA synthesis and the influence of UTP concentrations on the length of poly(U) sequences. (A) PV RF RNA fractionated by 1% agarose gel electrophoresis. PIRCs containing PV A₍₈₄₎ RNA templates (lanes 1 to 3) or PV A₍₈₄₎ KO CRE RNA templates (lanes 4 to 6) were incubated in reaction mixtures containing 1 mM ATP, 250 μM GTP, and 250 μM CTP, either 10 μM UTP (lanes 1, 3, 4, 6, and 7) or 100 μM UTP (lanes 2 and 5), and [α-³²P]UTP, with (lanes 3 and 6) or without 2 mM guanidine HCl, as described in “PV RNA replication” in Materials and Methods. Reaction products soluble in 2 M LiCl were separated by 1% agarose gel electrophoresis and detected by phosphorimaging. The mobility of PV RF RNA is indicated. (B) RNase T₁ oligonucleotides in PV RNAs. PV A₍₈₄₎ RNA templates (lane 1) and PV A₍₈₄₎ KO CRE RNA templates (lane 2) were synthesized by T7 RNA transcription in reaction mixtures containing [α-³²P]ATP (see Materials and Methods), digested with RNase T₁, and separated by electrophoresis in 7 M urea-20% polyacrylamide (see “RNase T₁ digestion” in Materials and Methods) (lanes 1 and 2). [α-³²P]UTP-radiolabeled products of PV A₍₈₄₎ RNA replication (lanes 3 to 5) or PV A₍₈₄₎ KO CRE RNA replication (lanes 6 to 8) from reaction mixtures corresponding to lanes 1 to 6 of panel A were digested with RNase T₁ and separated by electrophoresis in 7 M urea-18% polyacrylamide (see “PV RNA replication” and “RNase T₁ digestion” in Materials and Methods). The mobilities of specific T₁ oligonucleotides and VPg-linked poly(U) are indicated. A 120-base-long RNA, corresponding to the 5′ 120 bases of PV RNA, was used as a size marker in the urea-polyacrylamide gels. (C) Size distributions of poly(A) sequences of PV A₍₈₄₎ RNA templates and the corresponding VPg-linked poly(U) products of RNA replication. Amounts of RNase T₁ oligonucleotides from PV A₍₈₄₎ RNA templates (green line), VPg-linked poly(U) products from RNA replication reaction mixtures containing 10 μM UTP (red line), and VPg-linked poly(U) products from RNA replication reaction mixtures containing 100 μM UTP (blue line) are indicated. The molar amounts of VPg-linked poly(U) products were calculated based on the corresponding molar amounts of RNase T₁ oligonucleotides from the heteropolymeric portion of each RNA product. PI, phosphorimaging.