The Arabidopsis thaliana KAT1 cDNA encodes a voltage-gated inward-rectifying K+ channel. A KAT1 genomic DNA clone was isolated and sequenced, and a 5' promoter and coding sequences containing eight introns were identified. Reporter gene analysis of transgenic plants containing the KAT1 promoter fused to bacterial beta-glucuronidase showed robust beta-glucuronidase activity primarily in guard cells.

SUMMARY Overlapping genes pose an evolutionary dilemma as one DNA sequence evolves under the selection pressures of multiple proteins. Here, we perform systematic statistical and mutational analyses of the overlapping HIV-1 genes tat and rev and engineer exhaustive libraries of non-overlapped viruses to perform deep mutational scanning of each gene independently. We find a “segregated” organization in which overlapped sites encode functional residues of one gene or the other, but never both. Furthermore, this organization eliminates unfit genotypes, providing a fitness advantage to the population. Our comprehensive analysis reveals the extraordinary manner in which HIV minimizes the constraint of overlapping genes and repurposes that constraint to its own advantage. Thus, overlaps are not just consequences of evolutionary constraints, but rather can provide population fitness advantages.

Among the highly conserved sites in K ؉ channel pores, the tyrosine-glycine sequence is believed to play an important role in selectivity. Here we describe a novel approach in which comprehensive mutagenesis of the YG sites of the voltage-gated K ؉ channel, Kat1, is combined with phenotypic screening in Saccharomyces cerevisiae and electrophysiological analysis in Xenopus oocytes to determine the roles of these sites in K ؉ selectivity. We show that structural constraints necessitate a tyrosine or phenylalanine at the first position to confer full K ؉ selectivity. Substitution to arginine creates a channel titratable by external pH, suggesting that the side group at this position may line the channel pore. Permeation is abolished by any increase in bulk at the adjacent glycine position unless accompanied by a compensatory mutation at the tyrosine site. These results suggest a model in which the selectivity filter of the K ؉ channel requires an aromatic residue paired with glycine within the pore loop in order to maintain maximal K ؉ selectivity.

Human immunodeficiency virus type 1 (HIV-1) transcription is regulated by the viral Tat protein, which relieves a block to elongation by recruiting an elongation factor, P-TEFb, to the viral promoter. Here, we report the discovery of potent Tat inhibitors that utilize a localization signal to target a dominant negative protein to its site of action. Fusing the Tat activation domain to some splicing factors, particularly to the Arg-Ser (RS) domain of U2AF65, creates Tat inhibitors that localize to subnuclear speckles, sites where pre-mRNA processing factors are stored for assembly into transcription complexes. A U2AF65 fusion named T-RS interacts with the nonphosphorylated C-terminal domain of RNA polymerase II (RNAP II) via its RS domain and is loaded into RNAP II holoenzyme complexes. T-RS is recruited efficiently to the HIV-1 promoter in a TARindependent manner before RNAP II hyperphosphorylation but not to cellular promoters. The "preloading" of T-RS into HIV-1 preinitiation complexes prevents the entry of active Tat molecules, leaving the complexes in an elongation-incompetent state and effectively suppressing HIV-1 replication. The ability to deliver inhibitors to transcription complexes through the use of targeting/localization signals may provide new avenues for designing viral and transcription inhibitors.

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