Mapping of a DNA binding region of the PI-sceI homing endonuclease by affinity cleavage and alanine-scanning mutagenesis.

The PI-SceI protein is a member of the LAGLIDADG family of homing endonucleases that is generated by a protein splicing reaction. PI-SceI has a bipartite domain structure, and the protein splicing and endonucleolytic reactions are catalyzed by residues in domains I and II, respectively. Structural and mutational evidence indicates that both domains mediate DNA binding. Treatment of the protein with trypsin breaks a peptide bond within a disordered region of the endonuclease domain situated between residues Val-270 and Leu-280 and interferes with the ability of this domain to bind DNA. To identify specific residues in this region that are involved in DNA binding and/or catalysis, alanine-scanning mutagenesis was used to create a set of PI-SceI mutant proteins that were assayed for activity. One of these mutants, N281A, was >300-fold less active than wild-type PI-SceI, and two other proteins, R277A and N284A, were completely inactive. These decreases in cleavage activity parallel similar decreases in substrate binding by the endonuclease domains of these mutant proteins. We mapped the approximate position of the disordered region to one of the ends of the 31 base pair PI-SceI recognition sequence using mutant proteins that were substituted with cysteine at residues Asn-274 and Glu-283 and tethered to the chemical nuclease FeBABE. These mutational and affinity cleavage data strongly support a model of PI-SceI docked to its DNA substrate that suggests that one or more residues identified here are responsible for contacting base pair A/T(-)(9), which is essential for substrate binding.     

Mutational analysis of an extracytoplasmic-function sigma factor to investigate its interactions with RNA polymerase and DNA

The extracytoplasmic-function (ECF) family of sigma factors comprises a large group of proteins required for synthesis of a wide variety of extracytoplasmic products by bacteria. Residues important for core RNA polymerase (RNAP) binding, DNA melting, and promoter recognition have been identified in conserved regions 2 and 4.2 of primary sigma factors. Seventeen residues in region 2 and eight residues in region 4.2 of an ECF sigma factor, PvdS from Pseudomonas aeruginosa, were selected for alanine-scanning mutagenesis on the basis of sequence alignments with other sigma factors. Fourteen of the mutations in region 2 had a significant effect on protein function in an in vivo assay. Four proteins with alterations in regions 2.1 and 2.2 were purified as His-tagged fusions, and all showed a reduced affinity for core RNAP in vitro, consistent with a role in core binding. Region 2.3 and 2.4 mutant proteins retained the ability to bind core RNAP, but four mutants had reduced or no ability to cause core RNA polymerase to bind promoter DNA in a band-shift assay, identifying residues important for DNA binding. All mutations in region 4.2 reduced the activity of PvdS in vivo. Two of the region 4.2 mutant proteins were purified, and each showed a reduced ability to cause core RNA polymerase to bind to promoter DNA. The results show that some residues in PvdS have functions equivalent to those of corresponding residues in primary sigma factors; however, they also show that several residues not shared with primary sigma factors contribute to protein function.