Testing the role of predicted motifs -- an example


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:


REFERENCE:

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