Table 1.
Summary of the characterization of RNAP II mutants.
| RNAPII | In vitro assays
|
In vivo location
|
||||||
|---|---|---|---|---|---|---|---|---|
| RNAPII assembly | PIC formation | Initiation on premelted promoter | Initiation on closed promoter | NTP-Mg(B) binding | Transcript elongation | Promoter | Transcribed region | |
| Rpb2 | ||||||||
| WT | + | + | + | + | + | + | + | + |
| Switch3 (R1078A, S1079A, R1080A) | + | ± | − | − | − | − | − | |
| Fork 1 (Δ458–459) | + | + | ± | − | − | + | − | |
| Conserved E791A (E791A) | + | + | + | + | ± | ± | + | ± |
| Rpb1 | ||||||||
| Rudder (Δ321–334) | + | − | − | − | − | |||
| Zipper (Δ45–55) | + | − | − | − | − | |||
| WT | + | + | + | + | + | |||
Note: Various assays that monitor the different stages of the transcription reaction were used to compare each RNAP II mutant to the wild-type enzyme. Our assays monitored the following: 12-subunit core RNAP II assembly, preinitiation complex (PIC) formation on promoter DNA in the presence of the general transcription factors, abortive initiation on a premelted template (this assay allowed us to bypass the promoter melting requirement), abortive initiation on a closed template, NTP–Mg binding, and elongation using a C-tailed template or a promoter-dependent run off assay. ChIP assays were used to localize the RNAP II variants on the promoter or transcribed region of active genes in vivo. NTP, nucleoside triphosphate; empty boxes, not determined.