Study of T7 RNA polymerase-DNA interactions by way of unnatural base pairs
The use of the T7 RNA polymerase is ubiquitous in molecular biology as a research tool for the study of the regulation and mechanism of transcription in biological systems. Previous research has demonstrated its high promoter specificity; therefore, it is commonly used for transcription of DNA cloned into bacterial vectors1. This high promoter specificity also ensures that any modified or mutated promoter likely will cause the T7 RNA polymerase to bind inefficiently when encountering an altered base and cease the transcription of its DNA substrate. Especially critical are the base pairs at the -16 to +6 positions relative to the start site. Many of these base pairs are involved in the initial binding of the T7 RNA polymerase protein and the formation of the transcription bubble1. This experiment focused on the effects of altering these critical base pairs upstream of the transcription start site in conjunction with the modification of the T7 RNA polymerase to gain a better understanding of protein-DNA interactions and yield possible new techniques in research2. Specifically, the major groove interactions between the T7 RNA polymerase and bases in the -9 to -7 position relative to the start site were studied1. Wild-type bases were replaced with a combination of wild-type substitutions, iso-forms of wild-type bases, and artificial bases with modified functional groups. In this experiment, both iso-Guanine and iso-Cytosine pairs were substituted at multiple locations in the promoter. Furthermore, a completely unnatural base pair of dZ:dP was used for substitutions. These are unique due to the dP base missing an N7 group and an NO2 being added to the dZ base. In addition to these promoter alterations, multiple variants of the wild-type T7 RNA polymerase were produced by replacing amino acids responsible for the protein-DNA interactions seen in transcription. These variants were tested with modified promoters to ascertain the success of transcription. Within the time constraints of this project, moderate success was found when some variants increased the transcription activity normally seen between the modified promoters and the wild-type T7 RNA polymerase. Future studies in this field may include testing more variant T7 RNA polymerases and decoding details of DNA-protein interactions. Furthermore, this experiment extends the possibility of obtaining a modified promoter with a compatible T7 RNA Polymerase where both must concurrently be present for transcription to successfully occur. With this new research technique, single genes could be up-regulated or down-regulated with a high degree of accuracy due to the uniqueness of the promoter sequence. An unnatural promoter sequence with little tolerance of wild-type polymerases would ensure that no other genes are upregulated when using the matching modified T7 RNA polymerase.