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We recently proposed that a nontemplate strand base in the discriminator region of bacterial promoters, the region between the -10 element and the transcription start site, makes sequence-specific contacts to region 1.2 of the [sigma] subunit of Escherichia coli RNA polymerase (RNAP). Because rRNA promoters contain sequences within the discriminator region that are suboptimal for interaction with [sigma]1.2, these promoters have the kinetic properties required for regulation by the RNAP-binding factors DksA and ppGpp. Here, we use zero-length cross-linking and mutational, kinetic, and footprinting studies to map RNAP interactions with the nontemplate strand bases at the junction of the -10 element and the discriminator region in an unregulated rRNA promoter variant and in the [LAMBDA] [P.sub.R] promoter. Our studies indicate that nontemplate strand bases adjacent to the -10 element bind within a 9-aa interval in [sigma]1.2 (residues 99-107). We also demonstrate that the downstream-most base on the nontemplate strand of the -10 hexamer cross-links to [sigma] region 2, and not to [sigma]1.2. Our results refine models of RNAP-DNA interactions in the promoter complex that are crucial for regulation of transcription initiation. promoter element | RNA polymerase | transcription initiation | discriminator region | 10 element

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.04.034 Byline: Shanil P. Haugen (1), Melanie B. Berkmen (1), Wilma Ross (1), Tamas Gaal (1), Christopher Ward (1), Richard L. Gourse (1) Abstract: Regulation of transcription initiation is generally attributable to activator/repressor proteins that bind to specific DNA sequences. However, regulators can also achieve specificity by binding directly to RNA polymerase (RNAP) and exploiting the kinetic variation intrinsic to different RNAP-promoter complexes. We report here a previously unknown interaction with Escherichia coli RNAP that defines an additional recognition element in bacterial promoters. The strength of this sequence-specific interaction varies at different promoters and affects the lifetime of the complex with RNAP. Selection of rRNA promoter mutants forming long-lived complexes, kinetic analyses of duplex and bubble templates, dimethylsulfate footprinting, and zero-Angstrom crosslinking demonstrated that I subunit region 1.2 directly contacts the nontemplate strand base two positions downstream of the -10 element (within the 'discriminator' region). By making a nonoptimal I1.2-discriminator interaction, rRNA promoters create the short-lived complex required for specific responses to the RNAP binding factors ppGpp and DksA, ultimately accounting for regulation of ribosome synthesis. Author Affiliation: (1) Department of Bacteriology, University of Wisconsin-Madison, 420 Henry Mall, Madison, WI 53706, USA Article History: Received 14 January 2006; Revised 16 March 2006; Accepted 11 April 2006 Article Note: (miscellaneous) Published: June 15, 2006

A study reports an unknown interaction with Escherichia coli ribonucleic acid polymerase (RNAP) that defines an additional recognition element in bacterial promoters. Results indicate that ribosomal RNA (rRNA) promoters create the short-lived complex required for specific responses to the RNAP binding factors ppGpp and DksA by making a non-optimal sigma1.2-discriminator interaction ultimately accounting for regulation of ribosome synthesis.

We recently proposed that a nontemplate strand base in the discriminator region of bacterial promoters, the region between the -10 element and the transcription start site, makes sequence-specific contacts to region 1.2 of the r subunit of Escherichia coli RNA polymerase (RNAP). Because rRNA promoters contain sequences within the discriminator region that are suboptimal for interaction with ul .2, these promoters have the kinetic properties required for regulation by the RNAP-binding factors DksA and ppGpp. Here, we use zero-length cross-linking and mutational, kinetic, and footprinting studies to map RNAP interactions with the nontemplate strand bases atthe junction of the -10 element and the discriminator region in an unregulated rRNA promoter variant and in the APR promoter. Our studies indicate that nontemplate strand bases adjacent to the -10 element bind within a 9-aa interval in rn .2 (residues 99-107). We also demonstrate that the downstream-most base on the nontemplate strand of the -10 hexamer cross-links to rn region 2, and not to rnl.2. Our results refine models of RNAP-DNA interactions in the promoter complex that are crucial for regulation of transcription initiation. [ABSTRACT FROM AUTHOR]

Introduction. Simethicone is an over-the-counter product that is frequently used by clinicians during endoscopic procedures to reduce foaming and improve visualization. Published studies have found simethicone residue on endoscopes after cleaning and disinfecting the devices as per the manufacturer's instructions. Some literature suggests that simethicone residue may reduce disinfection efficacy and increase the risk of patient infections. Gap Statement. However, there appears to be a lack of direct evidence in the literature to either disprove this or correlate simethicone presence with an increased microbial risk. Aim : Research was conducted to evaluate the in vitro impact of simethicone on disinfection efficacy. Methodology. Bacteria were grown in a microtitre plate assay in the presence of a range of simethicone concentrations and then treated with a disinfectant. Bacterial growth was assessed by spotting each microtitre well onto an agar plate. Results. The results demonstrated that, under the conditions tested, simethicone did not reduce the efficacy of Cidex ortho-phthalaldehyde disinfectant, which demonstrated at least a 6-log unit reduction in bacterial viability. Additional experiments showed that direct exposure to 66 mg ml -1 of simethicone reduced bacterial viability. Conclusion. These results indicate that simethicone may not reduce the bactericidal efficacy of disinfectant during reprocessing, under certain conditions.