Fig. 7: C11 can activate C37 CTD-termination loop mutants for recycling.

a The NTD-L of C11 can activate C37-termination/initiation loop mutants for Pol III recycling. In vitro transcription reactions using a batch of SUP4-tRNA^Tyr complexes reconstituted with Pol III-core alone (lane 1), or Pol III-core preincubated with C37/53 or C37*/53 in which the five amino acids at positions 226–230 are substituted with alanine, or C37^D/53 in which they are deleted, alone (lanes 2–4), with C11 (WT, lanes 5–7) or with the NTD-L (lanes 8–10), as indicated. The T1 and T2 bands are as described for Fig. 4b and d. b, c Invariant linker mutations affect RNA 3′-cleavage activity but not Pol III recycling. The two C11 constructs with D52A,D53A mutations to the invariant central linker (Lkmt) are shown in Fig. 2a. b The RNA 3′-overhang substrate representing Pol III backtrack arrested ECs was incubated with either buffer alone, C11 or C11-(Lkmt) in amounts indicated above the lanes under standard assay conditions. Cleavage products (CP) are indicated. c Results of in vitro transcription reactions using a batch of SUP4-tRNA^Tyr complexes reconstituted with Pol III-core alone (lane 1) or aliquots of a batch of Pol III-core preincubated with C37/53 together with C11, NTD-L, or the linker-mutated versions thereof as indicated above the lanes (the misshapen band in lane 6 resulted from a similarly misshaped loading well in the gel). The T1 and T2 bands are as described for Fig. 4b and d.