Figure 1.

Assembly of SARS-CoV-2 RNA polymerase catalytic complexes

A schematic diagram of the catalytic complex assembly, purification, and reactivity assays of a stalled post-translocated complex (A) and an RDV-MP incorporated pre-translocated complex (B). Abbreviation is as follows: IEX: ion-exchange chromatography.

(A) The post-translocated complex assembly was performed by using a T33-1:P10 construct, the three nsp proteins, and CTP/ATP as the only NTP substrates to allow the synthesis of a 14-mer product (P14). The majority of the P10 was converted to P14 after a 90-min incubation (“M” indicates a marker with a mixture of 33-mer and 10-mer RNAs). The P14-containing catalytic complex was purified by anion exchange chromatography, and the purified complex can further react with GTP (G) or 3¢-deoxy-GTP (3dG) to yield the 16-mer (P16) or 15-mer containing complexes, respectively. The structure of this P14-containing complex was solved in cryo-EM trials at the post-translocated state.

(B) The pre-translocated complex was obtained through a two-step process. In the first step, a T33-7:P10 construct, the three nsp proteins, and CTP/ATP were mixed to allow the synthesis of a 14-mer product (P14). The majority of the P10 are converted to P14 after a 180-min incubation. The P14-containing catalytic complex was purified by anion exchange chromatography, and the purified complex can further react with G or 3dG to yield the 16-mer (P16) or 15-mer containing complexes, respectively. In the second step, the purified P14-containing complex was mixed with GTP and RDV-TP, and a pausing event corresponding to the synthesis of an 18-mer product (P18) was observed. The structure of this P18-containing complex was solved in cryo-EM trials at the pre-translocated state.

See also Figure S1.