: The synthesis of the translation apparatus is central to growth and development of all organisms, and its regulation has been a central issue in molecular microbioogy for over 50 years. Recently, it has become clear that an understanding of the mechanisms responsible for rRNA and tRNA transcription can provide fundamental insights into the mechanism of transcription in general. We determined in the previous project period that not only does the nucleotide derivative ppGpp directly regulate rRNA promoters, but it also directly regulates transcription much more widely than previously expected, with more than 700 transcripts changing either negatively or positively within 5 minutes of ppGpp induction. In the major aim of the proposal, we will analyze the regulation of transcription by ppGpp and its cofactor DksA. We found that there are two distinct binding sites for ppGpp on E. coli RNA polymerase (RNAP). We will determine the binding affinities of ppGpp for each binding site, determine whether binding is cooperative, develop and test mechanistic models for ppGpp action at each site including the mechanism of positive control, determine the regulatory roles of each ppGpp binding site on different promoters in vitro and in vivo, and address the roles of the two sites in regulation of transcription elongation. Finally, we will use metabolomic and proteomic approaches to identify ppGpp targets on proteins other than RNAP and investigate their significance. In the second aim, we will investigate the mechanisms of transcription factors that, like DksA, bind directly to RNAP. These include TraR and its homologs, distant DksA-like proteins carried on widely-distributed extrachromosomal elements; R. sphaeroides DksA-like and CarD-like proteins that appear to be important for regulation for responses to light; and E. coli Crl, a Sigma S holoenzyme assembly factor. We recently discovered that E. coli rRNA operons form a bacterial nucleolus-like structure that is not dependent on transcription but nevertheless requires the rRNA promoter region. In the third aim, we will continue to determine the cis- and trans-acting determnants for this structure and its consequences for cellular physiology.