Single-chain integration host factors as probes for high-precision nucleoprotein complex formation.
Integration host factor (IHF) is a heterodimeric, site-specific DNA-binding and DNA-bending protein from Escherichia coli. It is involved in high-precision DNA transactions where it serves as a key architectural component of specialized nucleoprotein structures (snups). We described recently a novel approach for protein engineering using a single polypeptide chain IHF, termed scIHF2, as a first example. ScIHF2 is made up of the alpha subunit of IHF which was inserted into the beta subunit at peptide bond Q39/G40 via two short linkers. The monomer behaves very similarly to the heterodimeric, parental IHF in biochemical and functional assays. Here, we describe an extension of this approach in which we shortened either one or both linkers by one amino acid, thereby generating three new variants termed scIHF1, 3, and 4. These variants exhibit distinct DNA-binding properties, different phenotypes in site-specific integrative and excisive recombination by phage lambda integrase in vitro, as well as in pSC101 replication assays in a DeltaIHF E. coli host. We also introduced a K45E substitution within the alpha domain of scIHF3 and based on electrophoretic mobility shift assays (EMSAs), argue that it significantly changes the DNA trajectory within the protein-DNA complex. Our results indicate that IHF's pleiotropic roles in DNA transactions inside E. coli require different types of high-precision DNA architectural activities. The scIHF variants described here will help to explore further how flexible these requirements are.
Bao
Qiuye
Q
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
Christ
Nicole
N
Dröge
Peter
P
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics; D017931:DNA Primers; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D034982:Integration Host Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011995:Recombination, Genetic; D017384:Sequence Deletion
D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D034982:Integration Host Factors