CD and DNA binding studies of a proline repeat-containing segment of the replication arrest protein Tus.
Tus is a sequence-specific DNA binding protein that regulates its own transcription and can arrest Escherichia coli replication when bound to Ter sites on the chromosome. In order to identify segments of Tus that may be involved in DNA binding interactions we have analyzed the Tus amino acid sequence with respect to secondary structure motifs and similarity to other protein sequences. A twenty amino acid segment containing several basic residues and a proline repeat motif with a periodicity of five residues was identified. The motif was common to several other nucleic acid binding proteins, including histone H1-3, Xenopus laevis ribosomal protein L1, and the single-stranded DNA binding protein (DBP) from adenovirus. A 22 amino acid peptide, TPPI, having a sequence similar to the Tus segment binds non-specifically and non-cooperatively to double- and single-stranded DNA with a binding constant of 1.5 +/- 0.2 x 10(6) M-1. The estimated binding site size was 4.3 +/- 0.5 base pairs. Circular dichroism studies indicated that the peptide was a random coil in buffer but adopted a helical structure in 50% trifluroethanol and in sodium dodecyl sulfate at concentrations above the critical micellar concentration. Several helical models of the TPPI sequence were constructed graphically and minimized. One of them, an amphiphilic, left-handed, 5.1(19) helical model was best able to account for the observed structural properties of TPPI in the presence of structure-promoting additives.
Nedved
M L
ML
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Gottlieb
P A
PA
Moe
G R
GR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D003198:Computer Simulation; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D029968:Escherichia coli Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis; D011089:Polydeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D011392:Proline / Q000235:genetics*; D017433:Protein Structure, Secondary; D012967:Sodium Dodecyl Sulfate; D014270:Trifluoroethanol
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D010455:Peptides; D011089:Polydeoxyribonucleotides; C061910:tus protein, Bacteria; C489236:tus protein, E coli; D012967:Sodium Dodecyl Sulfate; D014270:Trifluoroethanol; D004247:DNA; D011392:Proline