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.

The diversity of RNAs inside living cells is amazing. We have known of the more “classic” RNA species: mRNA, tRNA, rRNA, snRNA and snoRNA for some time now, but in a steady stream new types of molecules are being described as it is becoming clear that most of the genomic information of cells ends up in RNA. To deal with the enormous load of resulting RNA processing and degradation reactions, cells need adequate and efficient molecular machines. The RNA exosome is arising as a major facilitator to this effect. Structural and functional data gathered over the last decade have illustrated the biochemical importance of this multimeric complex and its many co-factors, revealing its enormous regulatory power. By gathering some of the most prominent researchers in the exosome field, it is the aim of this volume to introduce this fascinating protein complex as well as to give a timely and rich account of its many functions. The exosome was discovered more than a decade ago by Phil Mitchell and David Tollervey by its ability to trim the 3’end of yeast, S. cerevisiae, 5. 8S rRNA. In a historic account they laid out the events surrounding this identification and the subsequent birth of the research field. In the chapter by Kurt Januszyk and Christopher Lima the structural organization of eukaryotic exosomes and their evolutionary counterparts in bacteria and archaea are discussed in large part through presentation of structures.

This monograph reviews and summarizes the substantial body of work that has been published on the transcription by polymerase III over the past 5 years. Progress in this field has been very rapid since 1993, and this new edition incorporates all the recent developments and offers the reader a highly detailed analysis of the current state of research on this largest and most complex of the eukaryotic RNA polymerases.