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.

Vols. for 1963- include as pt. 2 of the Jan. issue: Medical subject headings.

The second edition explains the principles of recombinant DNA technology as well as other important techniques such as DNA sequencing, the polymerase chain reaction, and the production of monclonal antibodies.

This book provides a wide spectrum of methods to study RNA chaperones in vitro, at the single molecule level, and protocols useful for cell-based assays. Beginning with a section on a number of bacterial proteins for study, the volume also explores proteins from eukaryotic cells and how to delve into the complex interactions between RNA chaperones and the folding and unfolding of proteins. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, RNA Chaperones: Methods and Protocols serves as an ideal guide for scientists and students interested in RNA biology and RNA chaperones. Chapter 3 is available Open Access under a CC-BY 4.0 license via link.springer.com.

This book contains 14 original review chapters each yielding new, exciting and intriguing data about the emerging understanding of nucleolar structure and function in normal, stressed and diseased cells. The goal of this work is to provide special insight into the nucleolus of the past, present and future, as well its regulation, translocation, and biomedical function. A multitude of topics are introduced and discussed in detail, including nucleologenesis, nucleolar architecture, nucleolar targeting, retention, anchoring, translocation, and the relationship between the nucleolus and cancer. This book also brings together work from several different species, from human to Drosophila to Dictyostelium and other eukaryotic microbes. The final chapter summarizes some of the issues brought up in the various chapters with a view to future research. This book supports the continued emergence of the nucleolus as a dynamic intranuclear region that oversees a vast diversity of events.