Recognition sites of 3′-OH group by T7 RNA polymerase and its application to transcriptional sequencing

When analyzing the elongation mechanisms in T7 RNA polymerase (T7 RNAP)by using site-directed mutagenesis and a protein expression system, we identified the recognition sites of the rNTP 3'-OH group in T7 RNAP. On the basis of three-dimensional crystal structure analysis, we selected sind analyzed six candidate sites interacting with the 3'-OH group of rNTP in T7 RNAP. We found that the Phe-644 and Phe-667 sites are responsible for the high selectivity of T7 RNAP for rNTPs. Also, we constructed the protein mutations of these residues, F644Y and F667Y, which display a >200-fold higher affinity than the wild type for 3'-dNTPs, These findings indicate that the phenylalaine residues of 644 and 667 specifically interact with the 3'-OH group. Thus, these mutants, F644Y and F667Y, with incorporation of 3'-dNTP terminators, which is similar to native rNTPs, can offer low backgrounds and equal intensities of the sequencing ladders in our method, called "transcriptional sequencing.".