Scott Allen wanted to characterize the DNA-binding site in the PutA protein from Salmonella typhimurium. The full-length PutA protein has 1320 amino acids and null mutations located throughout the putA gene prevent DNA binding. Within the DNA sequence of the putA gene, there is a short region that is very similar to the "helix-turn-helix" DNA-binding motif found in many other well-characterized DNA-binding proteins. This motif is so common in DNA-binding proteins that when this sequence is seen in a known DNA-binding protein, it is often assumed to be the DNA-binding domain.
To determine if this potential helix-turn-helix DNA-binding domain is involved in DNA binding by the PutA protein, Scott did site-directed mutagenesis of this sequence as shown below (using the single letter codes for amino acids).
I P L I A E T G G M N A M I V D S S A L A HTHI A A HTHII
Based upon predictions from known "helix-turn-helix" DNA-binding sites, changes of the indicated residues to Ala should prevent DNA-binding. The first mutant constructed (HTH I) contained the single Val (V) to Ala (A) substitution. The second mutant constructed (HTH II) contained two substitutions: Asp (D) to Ala, and Ser (S) to Ala.
Neither of the site-directed mutants prevented DNA-binding by the mutant PutA proteins. Therefore, Scott concluded that this "helix-turn-helix" motif is NOT required for DNA-binding by PutA protein.
Some questions worth pondering:
ATT CCG TTG ATT GCG GAA ACC GGC GGT ATG AAC GCT ATG ATT GTC GAC TCT TCC GCG CTC
For an excellent reference on sequence motifs and protein structure and function, see: Branden, C., and J. Tooze. 1999. Introduction to Protein Structure, Second Edition. Garland Publishing, Inc., NY.
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Last modified July 15, 2002