Fig. 6.

Reversal of NELF-mediated transcription repression depends on CE but not on cap formation. (A) Western blot of K294A mutant CE and wild-type CE. (B) Guanylylation of CE. Wild-type and mutant CE were incubated in 20 μl of GTP-labeling buffer (25 mM Tris·HCl, pH 7.5/5 mM MgCl₂/0.5 mM DTT/10 μCi (1 Ci = 37 GBq) [α-³²P]GTP/0.1 μg of inorganic pyrophosphatase) at 37°C for 10 min and then analyzed by SDS/PAGE and autoradiography. (C) PICs were incubated with DSIF and NELF as in Fig. 5A and then chased with NTPs in the absence and presence of wild-type CE (12 and 120 ng, compare lanes 4-7), K294A inactive mutant CE (12 and 120 ng, compare lanes 8-10), or BSA as control (120 ng, compare lanes 11 and 12). Analysis by 7% PAGE/8 M urea is shown.