Initial studies have been performed on transcription factor 1 (TF1), a type II DNA binding protein (DBPII) which displays a novel binding mode characterized by two beta-ribbon""""""""arms"""""""" interacting with the DNA. Completed studies include the determination of the solution structure of wild-type TF1 and of a DNA sequence from the genome of phage SPO1, containing the unusual base hydroxymethyluracil (HmU), which has been shown to bind preferentially to TF1. The solution structure of TF1 exhibits similarities to the structure of HU (another DBP II), but also some differences which may explain the difference in binding specificities for these homologous proteins. The structure of the beta-ribbon arms in TF1 is more defined in TF1 than in HU. Investigations on G15/I32-TF1 (a TF1 with increased DNA affinity) are proposed, including comparison of the solution structures of the mutant and wild-type protein, and their complexes with HmU-DNA. This mutant also demonstrates increased affinity when binding to DNA with two loops nine base pairs apart, which has been attributed to increased flexibility of the DNA.
The aim of these investigations is to develop a theoretical understanding of the preference of TF1 for HmU-containing DNA and the role of DNA flexibility in binding. Additional transcription factors gp33 and gp45 will be studied. Gp33 is a phage-encoded RNA polymerase-binding protein. After the solution structure is determined, the conformation of gp33 upon binding RNA polymerase will be studied. Gp45 is a transcription enhancing protein that binds DNA and is proposed to interact with gp33. NOESY spectra will be used to identify intramolecular contacts and generate structural models. The models will help to improve the understanding of the course of viral infection. Homo- and heteronuclear NMR methods applied to single- and double-labeled proteins will form the basis of these investigations.
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