FIGURE 3.

GMP-primed transcript has diphosphate at the 5′-end, and its β-phosphate is derived from ATP. A, only the GMP-primed transcript can be labeled with [γ-³²P]ATP in in vitro transcription. L-A virions were incubated with UTP and [γ-³²P]ATP in the presence of 20 μm GMP (lane 2), GDP (lane 3), or GTP (lane 4) or in the absence of guanine nucleotides (lane 1) as control. The 16-nt transcript was separated in a 15% acrylamide gel and visualized by autoradiography. B, GMP-primed 16-nt transcripts described above were isolated from the gel and treated with S1 nuclease (lane 2, S1), RNA 5′-polyphosphatase (lane 3, RPPase), or bacterial alkaline phosphatase (lane 4, BAP) and analyzed on PEI cellulose with 1 m LiCl. Lane 1, no enzyme-treated 16-nt transcripts. Lane 5, [γ-³²P]ATP. An autoradiogram of the thin layer is shown. The positions of nonlabeled GTP, GDP, and GMP are shown by the dotted ovals. C, the 5′-end region of L-A transcript is shown. The asterisk indicates that the β-phosphate at the 5′ terminus is derived from the γ phosphate of ATP when transcription is primed with GMP.