Copurification of the Lac repressor with polyhistidine-tagged proteins in immobilized metal affinity chromatography.

One of the commonly used resins for immobilized metal affinity purification of polyhistidine-tagged recombinant proteins is TALON resin, a cobalt (II)--carboxymethylaspartate-based matrix linked to Sepharose CL-6B. Here, we show that TALON resin efficiently purifies the native form of Lac repressor, which represents the major contaminant when (His)(6)-tagged proteins are isolated from Escherichia coli host cells carrying the lacI(q) gene. Inspection of the crystal structure of the repressor suggests that three His residues (residues 163, 173, and 202) in each subunit of the tetramer are optimally spaced on an exposed face of the protein to allow interaction with Co(II). In addition to establishing a more efficient procedure for purification of the Lac repressor, these studies indicate that non-lacI(q)-based expression systems yield significantly purer preparations of recombinant polyhistidine-tagged proteins.

An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana.

ARGONAUTE4 (AGO4) and RNA polymerase IV (Pol IV) are required for DNA methylation guided by 24 nucleotide small interfering RNAs (siRNAs) in Arabidopsis thaliana. Here we show that AGO4 localizes to nucleolus-associated bodies along with the Pol IV subunit NRPD1b; the small nuclear RNA (snRNA) binding protein SmD3; and two markers of Cajal bodies, trimethylguanosine-capped snRNAs and the U2 snRNA binding protein U2B''. AGO4 interacts with the C-terminal domain of NRPD1b, and AGO4 protein stability depends on upstream factors that synthesize siRNAs. AGO4 is also found, along with the DNA methyltransferase DRM2, throughout the nucleus at presumed DNA methylation target sites. Cajal bodies are conserved sites for the maturation of ribonucleoprotein complexes. Our results suggest a function for Cajal bodies as a center for the assembly of an AGO4/NRPD1b/siRNA complex, facilitating its function in RNA-directed gene silencing at target loci.

Dynamic regulation of ARGONAUTE4 within multiple nuclear bodies in Arabidopsis thaliana.

DNA methylation directed by 24-nucleotide small RNAs involves the small RNA-binding protein ARGONAUTE4 (AGO4), and it was previously shown that AGO4 localizes to nucleolus-adjacent Cajal bodies, sites of snRNP complex maturation. Here we demonstrate that AGO4 also localizes to a second class of nuclear bodies, called AB-bodies, which are found immediately adjacent to condensed 45S ribosomal DNA (rDNA) sequences. AB-bodies also contain other proteins involved in RNA-directed DNA methylation including NRPD1b (a subunit of the RNA Polymerase IV complex, RNA PolIV), NRPD2 (a second subunit of this complex), and the DNA methyltransferase DRM2. These two classes of AGO4 bodies are structurally independent--disruption of one class does not affect the other--suggesting a dynamic regulation of AGO4 within two distinct nuclear compartments in Arabidopsis. Abolishing Cajal body formation in a coilin mutant reduced overall AGO4 protein levels, and coilin dicer-like3 double mutants showed a small decrease in DNA methylation beyond that seen in dicer-like3 single mutants, suggesting that Cajal bodies are required for a fully functioning DNA methylation system in Arabidopsis.