The Effect of Partial Promoter Sequences on Primer Labeling and De Novo Initiation by T7 RNA Polymerase

The bacteriophage T7 RNA polymerase (T7 RNAP) is the prototype of the family of single subunit RNA polymerases which includes the T3, SP6 and mitochondrial RNA polymerases. It is also the most well characterized enzyme of this family of polymerases. T7 RNAP is the primary choice in studying the mechanistic aspects of transcription and promoter evolution owing to its high specificity for its promoter, requirement of no additional cofactors, and high fidelity of initiation from a specific site in its promoter. Although the two groups of single subunit polymerases, bacteriophage and mitochondrial, display a remarkable structural conservation, they recognize quite dissimilar promoters. Functional domains involved in promoter recognition and transcription initiation have been well characterized by thorough mutational studies by systematic deletions within these domains. T7 RNAP recognizes a 23 nucleotide (nt) promoter which can be divided into several functional domains. T7 RNAP can recognize a range of promoter sequences which are closely related to this consensus sequence. The promoter-specific recognition is achieved by the 13 nucleotide promoter recognition region which extends from -5 to -17 and contains sites important for polymerase recognition and positioning.

Here, we have developed an in vitro transcription system to study the ability of T7 RNAP to use truncated promoters similar to mitochondrial promoters. In this system, we used oligonucleotides which are capable of forming intra- and inter-molecular base pairing to produce a recessed 3’end on an extended 5’ template. We then systematically deleted nucleotides from the 5’ end of the 20-nucleotide double stranded region on these oligonucleotides containing the T7 RNAP promoter to determine (1) If they would label at the 3’ end of the oligonucleotide, (2) Initiate template-dependent de novo transcription, (3) Initiate template-independent and promoter-dependent de novo transcription or, (4) Fail to incorporate label when supplied with T7 RNAP and a single radiolabeled ribonucleotide triphosphate, which can base pair with the first unpaired base in the 5’ extension of the template. Under this condition, when a complete or almost complete (20 to 16 nt) double stranded T7 RNAP promoter is present, small RNAs are produced through template-independent and promoter-dependent stuttering corresponding to abortive initiation, which is lost when supplied with a completely scrambled promoter sequence. When partial double stranded promoter sequences (10-12 nt) are provided, template dependent de novo initiation of RNA occurs but at a site different from the canonical +1 initiation site (-GGG). In situations where no promoter is present but the potential exists to form a transient duplex region with a recessed 3’ end, the T7 RNAP adds a templated nucleotide to the 3’ end (primer labeling). Understanding the mechanisms underlying these observations helps us to understand the roles played by promoter elements. The evolution of promoter sequences of the single subunit RNAPs and to use this as a technique to synthesize defined RNAs without 5’- sequence constraints are discussed.