NA polymerases (RNAPs) synthesize RNA using DNA or RNA as a template. Accurate RNA synthesis is essential for cellular functions and viral RNA replication, so RNAPs actively select the right nucleotides by probing for nucleobase and nucleosugar moieties. RNAPs transcribing cellular genomes are large multi-subunit enzymes, whereas mitochondrial genomes are transcribed by structurally distinct single-subunit RNAPs. Viral RNAPs from RNA viruses are distantly related to mitochondrial RNAPs, but they use RNA as a template. Nucleoside analogues that mimic the canonical ribonucleotide triphosphate substrates (rNTPs) can be used to inhibit the RNAPs of pathogens. The mechanism of substrate selection by all RNAPs needs to be studied in great mechanistic detail to optimize analogues for selective targeting. This thesis work elucidates the mechanisms of nucleosugar selection and transcriptional proofreading by multi-subunit RNAPs, and provides insights into the nucleobase selectivity mechanism by different RNAP structural families. First, we found that multi-subunit RNAPs differentiate nucleosugar in ribo- and deoxyribonucleoside triphosphates (2’dNTPs) by utilizing the invariant arginine residue. This residue promotes rNTP binding, but also disfavors 2’dNTP incorporation into the RNA by stabilizing the catalytically inert 2’-endo conformation of the nucleosugar. Second, we delineated the contributions of various regions of the active site for proofreading activity of multi-subunit RNAPs. Third, we evaluated the suitability of six nucleoside analogues as substrates for multisubunit, mitochondrial and viral RNAPs. These RNAPs utilized the nucleoside analogues with different efficiencies and specificity. Several nucleoside analogues acted as dual coders, mimicking more than one canonical nucleobase. In particular, our data suggests that formycin A is a potent dual coder that may induce mutations during viral RNA synthesis. Overall, our results highlight the differences in substrate selection by cellular, mitochondrial and viral RNAPs, providing valuable information for the design of medically relevant transcription inhibitors.