Amino acid residues in region 2 of 70 have been shown to play an important role in the strand separation step that is necessary for formation of the functional or open RNA polymerase-promoter complex. Here we present a comparison of the roles of basic and aromatic amino acids in the accomplishment of this process, using RNA polymerase bearing alanine substitutions for both types of amino acids in region 2. We determined the effects of the substitutions on the kinetics of open complex formation, as well as on the ability of the RNA polymerase to form complexes with singlestranded DNA, and with forked DNA duplexes carrying a single-stranded overhang consisting of bases in the ؊10 region. We concluded that two basic amino acids (Lys 414 and Lys 418 ) are important for promoter binding and demonstrated distinct roles, at a subsequent step, for two aromatic amino acids (Tyr 430 and Trp 433 ). It is likely that these four amino acids, which are close to each other in the structure of 70 , together are involved in the nucleation of the strand separation process.RNA synthesis in prokaryotes is carried out by a multisubunit RNA polymerase commonly referred to as the core enzyme (E).1 For promoter recognition, a sigma (initiation) factor is required; it interacts with the core polymerase to yield the holoenzyme (E), which is able to form an initiation-competent complex at promoter sequences in a multistep process involving conformational changes in both the protein and the DNA (1-3). A striking feature of such a complex is a region of strand separation that spans about 14 base pairs from the upstream edge of the conserved Ϫ10 promoter element to just beyond the start site of transcription initiation (4). It is thought that, kinetically, strand separation initiates in the Ϫ10 region and proceeds in a downstream direction. Measurement of the size and location of the region of strand separation as a function of temperature shows that at low temperatures a small single-stranded region can be detected that, as the temperature is increased, expands toward the start site (3, 5-7). In addition, the introduction of nicks and mismatches in the Ϫ10 region is more effective in the acceleration of open complex formation than if such distortions are introduced at a more downstream position (8, 9).The predominant sigma factor in Escherichia coli, which enables recognition of promoters of housekeeping genes, is referred to as 70 . Sequence comparison has shown that a large group of sigma factors shows significant homology to 70 . Four regions of sequence conservation have been identified, of which some have been subdivided to reflect the most extensive sequence conservation (10). A large body of data has implicated region 2.3 of the main sigma factors of E. coli, Bacillus subtilis, and other prokaryotes in the nucleation of the strand separation. This process eventually results in the formation of the active or open complex, possibly by facilitating base flipping of the highly conserved A at Ϫ11 of the nontemplate strand. The support...

Voltage-gated potassium (Kv) channels extend their functional repertoire by coassembling with MinK-related peptides (MiRPs). MinK slows the activation of channels formed with KCNQ1 α subunits to generate the voltage-dependent IKs channel in human heart; MiRP1 and MiRP2 remove the voltage dependence of KCNQ1 to generate potassium “leak” currents in gastrointestinal epithelia. Other Kv α subunits interact with MiRP1 and MiRP2 but without loss of voltage dependence; the mechanism for this disparity is unknown. Here, sequence alignments revealed that the voltage-sensing S4 domain of KCNQ1 bears lower net charge (+3) than that of any other eukaryotic voltage-gated ion channel. We therefore examined the role of KCNQ1 S4 charges in channel activation using alanine-scanning mutagenesis and two-electrode voltage clamp. Alanine replacement of R231, at the N-terminal side of S4, produced constitutive activation in homomeric KCNQ1 channels, a phenomenon not observed with previous single amino acid substitutions in S4 of other channels. Homomeric KCNQ4 channels were also made constitutively active by mutagenesis to mimic the S4 charge balance of R231A-KCNQ1. Loss of single S4 charges at positions R231 or R237 produced constitutively active MinK-KCNQ1 channels and increased the constitutively active component of MiRP2-KCNQ1 currents. Charge addition to the CO2H-terminal half of S4 eliminated constitutive activation in MiRP2-KCNQ1 channels, whereas removal of homologous charges from KCNQ4 S4 produced constitutively active MiRP2-KCNQ4 channels. The results demonstrate that the unique S4 charge paucity of KCNQ1 facilitates its unique conversion to a leak channel by ancillary subunits such as MiRP2.

KCNQ1 α subunits form functionally distinct potassium channels by coassembling with KCNE ancillary subunits MinK and MiRP2. MinK-KCNQ1 channels generate the slowly activating, voltage-dependent cardiac I Ks current. MiRP2-KCNQ1 channels form a constitutively active current in the colon. The structural basis for these contrasting channel properties, and the mechanisms of α subunit modulation by KCNE subunits, are not fully understood. Here, scanning mutagenesis located a tryptophan-tolerant region at positions 338-340 within the KCNQ1 pore-lining S6 domain, suggesting an exposed region possibly amenable to interaction with transmembrane ancillary subunits. This hypothesis was tested using concomitant mutagenesis in KCNQ1 and in the membrane-localized 'activation triplet' regions of MinK and MiRP2 to identify pairs of residues that interact to control KCNQ1 activation.

Delayed rectifier potassium current diversity and regulation are essential for signal processing and integration in neuronal circuits. Here, we investigated a neuronal role for MinK-related peptides (MiRPs), membrane-spanning modulatory subunits that generate phenotypic diversity in cardiac potassium channels. Native coimmunoprecipitation from rat brain membranes identified two novel potassium channel complexes, MiRP2-Kv2.1 and MiRP2-Kv3.1b. MiRP2 reduces the current density of both channels, slows Kv3.1b activation, and slows both activation and deactivation of Kv2.1. Altering native MiRP2 expression levels by RNAi gene silencing or cDNA transfection toggles the magnitude and kinetics of endogenous delayed rectifier currents in PC12 cells and hippocampal neurons. Computer simulations predict that the slower gating of Kv3.1b in complexes with MiRP2 will broaden action potentials and lower sustainable firing frequency. Thus, MiRP2, unlike other known neuronal beta subunits, provides a mechanism for influence over multiple delayed rectifier potassium currents in mammalian CNS via modulation of alpha subunits from structurally and kinetically distinct subfamilies.

No abstract