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.

The basal eukaryotic transcription machinery for protein coding genes is highly conserved from yeast to high eukaryotes. However, while human cells usually initiate at a single transcription start site approximately 30 bp downstream of a TATA element, Schizosaccharomyces pombe typically initiates at multiple sites 30-70 bp, and Saccharomyces cerevisiae 40 to 200 bp downstream of the TATA. The determinant factor(s) for the species specific initiation and the underlying mechanisms for the multiple far downstream start site utilization in yeast are not well understood. By swapping the highly purified transcription factors between S. pombe and S. cerevisiae reconstituted transcription systems, we confirmed previous observations that RNA polymerase II and/or the general transcription factor TFIIB determine the species-specific start site utilization patterns. Further genetic and biochemical assays of TFIIB chimeras indicated that RNAPII, but not TFIIB as previously proposed, determines the distinct initiation patterns not only between the two yeast systems but also between human and yeast systems. Bubble template initiation assays showed that there is an inverse correlation between the amount of negative charge in the TFIIB B-fingertip and the efficiency of the first phosphodiester bond formation. Moreover, biochemical studies indicate that multiple initiation steps, including first phosphodiester bond formation, and RNA:DNA hybrid stability determined initiation-to-elongation transition, could be modulated to regulate the far downstream start sites utilization in S. cerevisiae. A model for multiple far downstream transcription start sites formation in S. cerevisiae is proposed.

Molecular diagnostic procedures have been described in a number of recent books and articles. However, these publications have not focused on virus detection, nor have they provided practical protocols for the newer molecular methods. Written by the inventors or principal developers of these technologies, Molecular Methods for Virus Detection provides both reviews of individual methods and instructions for detecting virus nucleic acid sequences in clinical specimens. Each procedure includes quality assurance protocols that are often ignored by other methodology books. Molecular Methods for Virus Detection provides clinically relevant procedures for many of the newer diagnostic methodologies. Provides state-of-the-art PCR methods for amplification, quantitation, in situ hybridization, and multiplex reactions Goes beyond PCR with protocols for 3SR, NASBA, LCR, SDA, and LAT Covers important virus detection methods such as in situ hybridization; Southern, dot, and slot blots; branched chain signal amplification; and chemiluminescence Includes quality control information crucial in research and clinical laboratories Most chapters are written by the inventors and principal developers of the methodologies Includes color plates, 77 figures, and 18 tables