DNA-mediated assembly of weakly interacting DNA-binding protein subunits: in vitro recruitment of phage 434 repressor and yeast GCN4 DNA-binding domains.
The specificity of DNA-mediated protein assembly was studied in two in vitro systems, based on (i) the DNA-binding domain of bacteriophage 434 repressor cI (amino acid residues 1-69), or (ii) the DNA-binding domain of the yeast transcription factor GCN4, (amino acids 1-34) and their respective oligonucleotide cognates. In vivo, both of these peptides are part of larger protein molecules that also contain dimerization domains, and the resulting dimers recognize cognate palindromic DNA sequences that contain two half-sites of 4 bp each. The dimerization domains were not included in the peptides tested, so in solution-in the presence or absence of non-cognate DNA oligonucleotides-these molecules did not show appreciable dimerization, as determined by pyrene excimer fluorescence spectroscopy and oxidative cross-linking monitored by mass spectrometry. Oligonucleotides with only one 4 bp cognate half-site were able to initiate measurable dimerization, and two half-sites were able to select specific dimers even from a heterogeneous pool of molecules of closely related specificity (such as DNA-binding domains of the 434 repressor and their engineered mutants that mimic the binding helix of the related P22 phage repressor). The fluorescent technique allowed us to separately monitor the unspecific, ionic interaction of the peptides with DNA which produced a roughly similar signal in the case of both cognate and non-cognate oligonucleotides. But in the former case, a concomitant excimer fluorescence signal showed the formation of correctly positioned dimers. The results suggest that DNA acts as a highly specific template for the recruitment of weakly interacting protein molecules that can thus build up highly specific complexes.
Guarnaccia
Corrado
C
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy.
Raman
Bakthisaran
B
Zahariev
Sotir
S
Simoncsits
András
A
Pongor
Sándor
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011721:Pyrenes / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D004220:Disulfides; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D021122:Protein Subunits; D011721:Pyrenes; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA; C030984:pyrene; D011494:Protein Kinases