The Esigma70-dependent N25 promoter is rate-limited at promoter escape. Here, RNA polymerase repeatedly initiates and aborts transcription, giving rise to a ladder of short RNAs 2-11 nucleotides long. Certain mutations in the initial transcribed sequence (ITS) of N25 lengthen the abortive initiation program, resulting in the release of very long abortive transcripts (VLATs) 16-19 nucleotides long. This phenomenon is completely dependent on sequences within the first 20 bases of the ITS since altering sequences downstream of +20 has no effect on their formation. VLAT formation also requires strong interactions between RNA polymerase and the promoter. Mutations that change the -35 and -10 hexamers and the intervening 17 base pair spacer away from consensus decrease the probability of aborting at positions +16 to +19. An unusual characteristic of the VLATs is their undiminished levels in the presence of GreB, which rescues abortive RNAs (

Calcineurin is responsible for mediating a wide variety of cellular processes in response to dynamic calcium (Ca 2+ ) signals, yet the precise mechanisms involved in the spatiotemporal control of calcineurin signaling are poorly understood. Here, we use genetically encoded fluorescent biosensors to directly probe the role of cytosolic Ca 2+ oscillations in modulating calcineurin activity dynamics in insulin-secreting MIN6 β-cells. We show that Ca 2+ oscillations induce distinct temporal patterns of calcineurin activity in the cytosol and plasma membrane vs at the ER and mitochondria in these cells. Furthermore, we found that these differential calcineurin activity patterns are determined by variations in the subcellular distribution of calmodulin (CaM), indicating that CaM plays an active role in shaping both the spatial and temporal aspects of calcineurin signaling. Together, our findings provide new insights into the mechanisms by which oscillatory signals are decoded to generate specific functional outputs within different cellular compartments.

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The efficiency of promoter escape in E. coli transcription initiation is dependent on two factors: 1) the rate of escape, and 2) the extent of RNA polymerase (RNAP) partitioning into escape‐competent open complexes. Both parameters can be obtained from a single cycle assay performed under RNAP‐limiting conditions. For T5 N25 and N25anti promoters, these two factors are governed by the initial transcribed sequence (ITS). N25 and N25anti promoters differ only in +3 to +20 region of the ITS, but exhibit half‐lives of ∼3 min and ∼42 min, respectively, for full‐length RNA synthesis. Interestingly, the slower escaping N25anti promoter forms a higher fraction of productive open complexes and yields higher levels of productive RNA than N25; this result was unexpected. The above studies were performed in 200 mM KCl solution. We next examined the effect of KCl concentration and found that lowering [KCl] from 200 – 10 mM increased the rate of escape for N25 while also raising its productive fraction. For N25anti, the escape rate remained the same while lower [KCl] brought about higher productive synthesis. We conclude that KCl concentration affects the open complex formation and partitioning similarly in both promoters, but influences their open complex collapse and escape differently, confirming that the rate of promoter escape is ITS‐sequence dependent. Supported by an NSF grant (RUI‐0418316) to L. M. Hsu.