The role of surface-exposed lysines in wrapping DNA about the bacterial histone-like protein HU.
Several basic proteins, including the ubiquitous HU proteins, serve histone-like functions in prokaryotes. Significant sequence conservation exists between HU homologues; yet binding sites varying from 9 to 37 bp have been reported. TF1, an HU homologue with a 37 bp binding site that is encoded by the Bacillus subtilis bacteriophage SPO1, binds with nM affinity to DNA that contains 5-hydroxymethyluracil (hmU) in place of thymine and to T-containing DNA with loops. We evaluated the contribution of three conserved lysines to specifying the length of the binding site and show that Lys3 is critical for maintaining a long binding site in T-containing DNA: A mutant protein in which Lys3 is replaced with Gln(TF1-K3Q) is completely deficient in forming a stable complex. The affinity for 37 bp hmU-containing DNA is also reduced, from approximately 3 nM for wild-type TF1 to approximately 90 nM for TF1-K3Q. The decrease in affinity of TF1-K3Q for hmU-containing DNA > or = 25 bp suggests that Lys3 contacts DNA 8-9 bp distal to the sites of kinking. We propose that Lys3 forms an internal saltbridge to Asp26 in HU homologues characterized by shorter binding sites and that its surface exposure, and hence a longer binding site, may correlate with absence of this aspartate.
Grove
Anne
A
Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, USA. agroves@lsu.edu
Saavedra
Tatiana C
TC
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D017103:Bacillus Phages / Q000737:chemistry; D001412:Bacillus subtilis / Q000821:virology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010432:Pentoxyl / Q000031:analogs & derivatives* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013941:Thymine / Q000378:metabolism; D014764:Viral Proteins
D001426:Bacterial Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D006657:Histones; C059361:TF1 DNA-binding protein, Bacteriophage SPO1; D014764:Viral Proteins; C040330:histone-like protein HU, bacteria; D001224:Aspartic Acid; C008055:5-hydroxymethyluracil; D010432:Pentoxyl; D008239:Lysine; D013941:Thymine