FIGURE 4.

Transcription initiation with 5′- and N6-amino derivatives of adenosine nucleotides under the T7 φ2.5 promoter (top) and their posttranscriptional derivatization with fluorescein (bottom). RNA samples were run to single-nucleotide resolution on an 8% denaturing PAGE and visualized by phosphorimaging. Top panel: Transcription in the presence (4 mM) of one of the 12 amino derivatives produces high yields of 5′ end amino-modified RNAs, with migration rates ranging from slightly below that of the N+1 band to that of the N+2 band of unmodified RNA, depending on the linker size. Bottom panel: Coupling of fluorescein with 5′ end amino-modified RNAs further slows down the RNA migration in the gel by approximately 1 nt (i.e., with migration rates varying from slightly below that of the N+2 band to that of the N+3 band of unmodified RNA). Both amino-RNA yields and fluorescein-labeled RNA yields are indicated under the corresponding gels. The yields of fluorescein-linked RNA agree well with the results of amino-RNA in the top panel.