Table II.
Sc RNAP II mutant proteins. Abbreviations: Gal (galactose), Glu (glucose), cs (cold sensitive), 6AU (6-azauracil), 5FOA (5-fluoro-orotic acid; counter-selection against URA3 plasmid carrying RPB2 wild type), KO (knockout), Purif (successful purification), Tx (transcription elongation; +++ represents wild type activity; efs (efs) indicates a strong (weak) defect in escape from a transcriptional stall).
| Sc Pol II | Tt RNAP | Region | Rationale | Gal | Glu | 5FOA | 6AU + Glu | KO TFIIS + Glu | Purif | Tx |
|---|---|---|---|---|---|---|---|---|---|---|
| Rpb2 R504A | β R420 | Fork Loop 2 | i+2 NTP interaction? | + | + | + | + | + | + | +++ |
| Rpb2 D505A | β E421 | Fork Loop 2 | i+2 NTP interaction? | + | + | +/− | + | + | + | ++ (efs) |
| Rpb2 K510A | β D426 | Fork Loop 2 | i+2 NTP interaction? | + | + | + | + | + | + | ++ |
| Rpb2 R512A | β R428 | Fork | Rpb2 R512C (forward genetics) | + | +/− cs | − cs | − | +/− | + | + |
| Rpb2 R512C* | β R428 | Fork | Rpb2 R512C (forward genetics) | + | + cs | − cs | − | +/− | + | + |
| Rpb2 Q513A | β D429 | Fork | i+2 NTP interaction? | + | + | + | + | + | + | +++ (efs) |
| Rpb2 E529A | β E445 | Fork/ Active Site | interaction with i+1 NTP? | + | + | +/− | +/− | +/− | + | ++++ |
| Rpb2 E529Q | β E445 | Fork/ Active Site | interaction with i+1 NTP? | + | + | +/− | +/− | +/− | + | ++ |
| Rpb2 E529D | β E445 | Fork/ Active Site | interaction with i+1 NTP? | + | + | + | + | + | + | ++++ (efs) |
| Rpb2 P528D G530A | β P444 G446 | Fork/ Active Site | alter flexibility of Fork | + | + | + | + | + | + | +++ |
| Rpb2 K979Q | β K838 | Active Site | i-site RNA phosphate interaction | + | − | − | − | − | n.d | n.d. |
| Rpb2 K979R | β K838 | Active Site | i-site RNA phosphate interaction | + | +/− | − | − | − | n.d | n.d. |
| Rpb2 G985A G988A | β G844 G847 | Active Site | alter flexibility of Active Site loop | + | + | + | + | + | + | +++ efs |
| Rpb2 R983Q | β R842 | Active Site | Rpb2 R983G (forward genetics) | + | + | + | +/− | + | + | ++ efs |
| Rpb2 K987Q | β K846 | Active Site | i-site RNA phosphate interaction | + | − | − | − | − | n.d | n.d. |
| Rpb2 K987R | β K846 | Active Site | i-site RNA phosphate interaction | + | − | − | − | − | n.d. | n.d. |
| Rpb2 S1019F | β S878 | Active Site | adjacent to Rpb2R1020 | + | + | + | − | + | + | + |
| Rpb2 R1020K | β R879 | Active Site | i+1 NTP γ-phosphate interaction | + | − | − | − | − | n.d. | n.d. |
| Rpb2 R1020Q | β R879 | Active Site | i+1 NTP γ-phosphate interaction | + | − | − | − | − | n.d. | n.d. |
| Rpb2 E1028Q | β E887 | Active Site | ion pair to Rpb2 R983 | + | + | +/− | + | +/− | + | +++ efs |
| wt | positive Control | + | + | + | + | + | + | +++ |