Key Roles of the Downstream Mobile Jaw of <i>Escherichia coli</i> RNA Polymerase in Transcription Initiation

Differences in kinetics of transcription initiation by RNA polymerase (RNAP) at different promoters tailor the pattern of gene expression to cellular needs. After initial binding, large conformational changes occur in promoter DNA and RNAP to form initiation-capable complexes. To understand the mechanism and regulation of transcription initiation, the nature and sequence of these conformational changes must be determined. <i>Escherichia coli</i> RNAP uses binding free energy to unwind and separate 13 base pairs of λP_R promoter DNA to form the unstable open intermediate I₂, which rapidly converts to much more stable open complexes (I₃, RP_o). Conversion of I₂ to RP_o involves folding/assembly of several mobile RNAP domains on downstream duplex DNA. Here, we investigate effects of a 42-residue deletion in the mobile β′ jaw (ΔJAW) and truncation of promoter DNA beyond +12 (DT+12) on the steps of initiation. We find that in stable ΔJAW open complexes the downstream boundary of hydroxyl radical protection shortens by 5–10 base pairs, as compared to wild-type (WT) complexes. Dissociation kinetics of open complexes formed with ΔJAW RNAP and/or DT+12 DNA resemble those deduced for the structurally uncharacterized intermediate I₃. Overall rate constants (<i>k</i>_a) for promoter binding and DNA opening by ΔJAW RNAP are much smaller than for WT RNAP. Values of <i>k</i>_a for WT RNAP with DT+12 and full-length λP_R are similar, though contributions of binding and isomerization steps differ. Hence, the jaw plays major roles both early and late in RP_o formation, while downstream DNA functions primarily as the assembly platform after DNA opening

Supporting workflow in a course management system

CMS, a secure and scalable web-based course management system developed by the Cornell University Computer Science Department, helps manage the workflow associated with running a course. Our goal in designing the system was to simplify, streamline, and automate all workflow aspects, such as course creation and importing students into it, student group management, assignment submission, assignment of graders, grading, regrade requests, and preparation of final grades. In contrast, other course management systems that we are aware of provide only specialized solutions for specific components, such as grading. This system is increasingly widely used for course management at Cornell University. The system was designed to support large courses with low administrative overhead. In this paper we describe the design of the system and the features that were found to be useful, and articulate its design principles