Mäkinen, J. J.; Belogurov, G. A.

RNA polymerase (RNAP) mediates the synthesis of an RNA copy of the template DNA, the first and often decisive step in gene expression. All cellular RNAPs possess an additional capacity to cleave nucleotides from the 3\' end of the nascent RNA. This ability potentially enhances the efficiency and accuracy of transcription, enabling RNAP to maintain processivity and ensure the fidelity of the RNA transcript. This study investigates the contributions of various active site regions to the RNA cleavage activity using a large collection of Escherichia coli RNAP variants. Unlike previous studies conducted under non-physiological conditions, this research employed backtracked RNAP complexes that cleave nascent RNA on a timescale of minutes under physiological pH and low Mg2+ concentrations. Our findings provide key insights into the RNA cleavage activity of the RNAP active site. Complete closure of the active site by the Trigger Loop (TL) facilitates RNA cleavage in 1-nt backtracked states, but not in 2-nt backtracked states. However, the RNA-proximal N-terminus of the TL influences the cleavage rate in both states. {beta} subunit Asp814 plays an important role in RNA cleavage, regardless of backtracking depth, likely by coordinating the Mg2+ ion responsible for generating the nucleophile. During RNA cleavage, the pre-translocated RNA nucleotide is base-paired to the template DNA, but its sugar-phosphate backbone is shifted compared to canonical pre-translocated and NTP-bound states. Bulky substitutions in the E-site (NTP entry area) stimulate RNA cleavage, suggesting that RNA binding in this site inhibits the reaction.