Abstract
In this study, several RNA polymerases were used for the first time to examine the possibility of transcriptional incorporation of 5’-N-triphosphates of 5’-amino-5’-deoxyribonucleosides (5’NH NTPs). The T3, T7, Sp6 and T7 Y639F RNA polymerases were employed to show that the full-length transcript cannot be synthesized. The results suggest that the application of 5’NH NTPs could decrease transcription reaction rates. What is more, the modification of transcription conditions had no influence on the rate of 5’NH NTPs incorporation. Based on experimental data it is postulated that 5’NH NTPs can be used as potential transcription inhibitors. Our findings expand the knowledge on suitable uses of the 5’-N-triphosphates of 5’-amino-5’-deoxyribonucleoside and the exact mechanism of transcriptional inhibition.
Highlights
During transcription, DNA-dependent RNA polymerases incorporate 5’-O-triphosphates of ribonucleosides into newly synthesized RNA
We tested the ability of four bacteriophage RNA polymerases: T7 RNA polymerase (T7), T7 Y693F mutant, T3 RNA polymerase (T3) and Sp6 RNA polymerase (Sp6) to incorporate 5’NH 5’-N-triphosphates of 5’amino-5’-deoxyribonucleosides (NTPs) into a transcript
When the transcription reaction was conducted with the Sp6 RNA polymerase, the intermediate product was detected only in the 5’NH GTP mixture (Fig 1, line 19)
Summary
Several RNA polymerases were used for the first time to examine the possibility of transcriptional incorporation of 5’-N-triphosphates of 5’-amino-5’-deoxyribonucleosides (5’NH NTPs). The results suggest that the application of 5’NH NTPs could decrease transcription reaction rates. The modification of transcription conditions had no influence on the rate of 5’NH NTPs incorporation. Our findings expand the knowledge on suitable uses of the 5’-N-triphosphates of 5’-amino-5’-deoxyribonucleoside and the exact mechanism of transcriptional inhibition. This publication was supported by the Polish Ministry of Science and Higher Education, under the KNOW program
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