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 and 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 phenylalanine 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. "
Izawa
M
M
Genome Science Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Koyadai 3-1-1, Tsukuba-city, Ibaraki 305, Japan.
Sasaki
N
N
Watahiki
M
M
Ohara
E
E
Yoneda
Y
Y
Muramatsu
M
M
Okazaki
Y
Y
Hayashizaki
Y
Y
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D012265:Ribonucleotides / Q000378:metabolism; D017421:Sequence Analysis / Q000379:methods; D014158:Transcription, Genetic / Q000235:genetics*; D014764:Viral Proteins
D012265:Ribonucleotides; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases