A systematic analysis was carried out to examine the effectes of ribonucleotide substitution at various locations within the promoter elemtn for T7 RNA polymerase. Ribonucleotides would be introduced at most positions without significantly decreasing transcription efficiency. A critical window of residues that wereinloerant of RNA substitution was defined for both the non-template and template strands of the promoter. Thee residues are involved in important contacts with the AT-rich recognition loop, specificity loop, and B-intercalating hairpin of the polymerase. These results highlight the malleability of T7 RNA polymerase in recognizing itspromoter element and suggest that promoters with altered backbone conformations may be used in molecular biology applications that us T7 RNA polymerase for in vitro transcription.