Intrinsic termination of T7 RNA polymerase transcription occurs at different signals in vitro. One type of signal is similar to that mediating factor‐independent termination of Escherichia coli RNA polymerase, whereas the other type does not involve RNA hairpin formation. By examining the termination behaviour of T7 RNA polymerase at the E.coli rrnB operon t1 terminator, at the T7‐t(phi) terminator, at the human preproparathyroid hormone gene terminator on both single‐ and double‐stranded templates, and in the presence of GTP or ITP during transcription, we show that the termination event can be mediated by either RNA or DNA structural features. Moreover, by using co‐transcriptional probing with potassium permanganate, we present evidence for the presence of transcription‐induced hyperreactive thymidines on the non‐template strand in the DNA‐mediated event, and a putative sequence motif is identified. We conclude that intrinsic termination of T7 RNA polymerase transcription in vitro can be mediated either by a hairpin in the nascent RNA or by a sequence motif including hyperreactive thymidines in the non‐template DNA strand.

ZP123 has no effects on atrial conduction during physiological conditions, but it selectively prevents atrial conduction slowing during metabolic stress.

Abstract:The ␥-aminobutyric acid (GABA) binding pocket within the GABA A receptor complex has been suggested to contain arginine residues. The aim of this study was to test this hypothesis by mutating arginine residues potentially contributing to the formation of a GABA binding pocket. Thus, six arginines conserved in human GABA A receptor ␣ subunits (arginine 34, 70, 77, 123, 135, and 224) as well as two nonconserved arginines (79 and 190), all located in the extracellular N-terminal segment of the ␣ 5 subunit, were substituted by lysines. The individual ␣ 5 subunit mutants were coexpressed with human ␤ 2 and ␥ 2s GABA A receptor subunits in Chinese hamster ovary cells by transient transfection. Electrophysiological whole-cell patch-clamp recordings show that, of the eight arginine residues tested, the two arginines at positions 70 and 123 appear to be essential for the GABA-gated chloride current because the EC 50 values of the two mutant constructs increase Ͼ100-fold compared with the wild-type ␣ 5 ,␤ 2 ,␥ 2s GABA A receptor. However, diazepam and allopregnanolone modulation and pentobarbital stimulation properties are unaffected by the introduction of lysines at positions 70 and 123. A double mutant carrying lysine substitutions at positions 70 and 123 is virtually insensitive to GABA, suggesting alterations of one or more GABA binding sites.

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