Fig. 5.

Tat/TAR-dependent phosphorylation of the RNAP II CTD stimulates cotranscriptional capping of viral mRNA. (A) HCE is recruited into HIV-1 TECs through phosphorylated RNAP II CTD. PICs were analyzed by Western blotting with α-CE antibody. TECs stalled at G36 and C81 were incubated with HCE and extensively washed to remove unbound proteins before Western blot analysis with α-CE antibody. The WT and TAR mutant (TM26) HIV-1 LTR templates are indicated. IN, input. (B) Tat/TAR-dependent phosphorylation of the RNAP II CTD stimulates capping of viral mRNA. The indicated TECs were supplemented with HCE and then incubated with [α-³²P]GTP. GMP-labeled transcripts were analyzed by electrophoresis through 10% polyacrylamide gels containing 7 M urea in TBE buffer followed by autoradiography (Upper). GMP-labeled RNAs isolated from the indicated TECs were analyzed by S1 nuclease digestion followed by polyethyleneimine-cellulose TLC (Lower).