Figure 2. Function of RbpA and RbpA derivatives in transcription initiation and cooperativity with CarD.
(A) Schematic diagram denoting the RbpA derivatives used in subsequent assays. (B) The effect of RbpA or RbpA derivatives (denoted at the bottom) on activation of abortive initiation from three different promoters (VapB, AP3, AP3anti-35, denoted at the top) with or without CarD. The transcription activity for each promoter was normalized with respect to holo activity on that promoter (holo alone on each promoter was normalized to a value of 1, as shown in lane 1). The error bars denote the standard error from a minimum of three experiments. (C) Promoter complex lifetimes measured by abortive initiation on the AP3 promoter. In the top panel, [32P]-labeled abortive transcript production at times after addition of a large excess of competitor promoter trap DNA (Davis et al., 2015) was monitored by polyacrylamide gel electrophoresis and autoradiography. On the bottom, transcript production was quantified by phosphorimagery and plotted. The lines indicate single-exponential decay curves fit to the data points. The calculated decay half-lives (t1/2) are shown to the right of the gel images. (D) Structural model showing the Msm RbpA/TIC (color coded as in Figure 1) along with CarD (red), superimposed by aligning the thermus CarD/RPo structure (PDB ID 4XLR; (Bae et al., 2015a).
Figure 2—figure supplement 1. Transcription templates and abortive initiation data.
