Co-transcriptional RNA proofreading by RNA polymerase (RNAP) is essential for accurate mRNA synthesis and reactivation of stalled transcription complexes, which can otherwise compromise genome integrity. RNAP from the stress-resistant bacterium Deinococcus radiodurans exhibits high levels of RNA cleavage in comparison with RNAP from Escherichia coli, which allows it to remove misincorporated nucleotides with high efficiency. Here, we show that the rate of RNA cleavage by D. radiodurans RNAP depends on the structure of the (mis)matched RNA 3'-nucleotide and its contacts with the active site. These interactions likely position the reactive phosphodiester bond in the cleavage-competent conformation, thus facilitating its hydrolysis catalyzed by metal ions in the active center. The universal RNA cleavage factor GreA largely alleviates defects in RNA cleavage caused by modifications in the RNA 3'-nucleotide or in its binding pocket in RNAP, suggesting that GreA functionally substitutes for these contacts. The results demonstrate that various RNAPs rely on a conserved mechanism for RNA proofreading, which can be modulated by changes in accessory parts of the active center. (C) 2018 Published by Elsevier Inc.