Book of Abstracts: Albany 2011

category image Albany 2011
Conversation 17
June 14-18 2011
©Adenine Press (2010)

Indirect Readout by the P22 Repressor Protein: How Charge and Solvent Organization Aid Binding Site Discrimination

To form a lysogen, the repressor of bacteriophage P22 (P22R) must bind and discriminate between its six DNA binding sites on the P22 chromosome. Comparison of the six naturally occurring binding site sequences shows that the sequences of the symmetrically-arrayed outermost base pairs in these sites are highly conserved, but the sequence of the innermost bases are not (Figure 1). Crystallographic studies show the conserved bases are directly contacted by bound protein, but the non-conserved central bases are not (1). Nonetheless through a sequence recognition strategy called “indirect readout”, the affinity of P22R for DNA varies with the non-contacted base sequence (2). We wish to understand the mechanism of indirect readout.

Figure 1: DNA sequences of naturally occuring operators in the P22 chromosome. The contacted and conserved bases are boxed and in bold. The central non-contacted bases are positions 8-11.

P22R DNA binding causes the non-contacted region to assume a B′-DNA configuration (1). The minor groove of B′-DNA is very narrow and contains a spine of highly ordered solvent molecules (1). We hypothesize that indirect readout depends on P22R’s ability to either sense and/or impose B′ structure on the bases in the non-contacted of its binding site. Thus the ease with which the non-contacted bases can assume B′ configuration by P22R determines the protein’s affinity for the binding site.

We showed that P22R binding sites lacking a minor groove N2-NH2 group on the base pairs at the non-contacted positions bind P22R ~12-fold better than those that do not contain this group at these positions. This finding identifies the N2-NH2 group as the mediator of indirect readout by P22R. We also found that changing either E44 or E48, which are directly juxtaposed to the DNA phosphate backbone near the non-contacted bases (1) (Figure 2), to uncharged residues eliminates the ability of P22R to recognize non-contacted base sequence. This finding suggests that these residues function to force the P22R-bound DNA into the B′ configuration. An N46A mutation completely blocks P22R’s ability to bind to sites bearing sequences in the non-contacted region, which cannot normally assume the B′ configuration. This finding suggests that N46 is responsible for inducing and/or stabilizing B′ conformation in the non-contacted bases in P22R-DNA complexes.

Figure 2: Positions of E44 and E48 relative to the DNA backbone. The P22R protein is above and the DNA is below. E44 and E48 are spacefilled (yellow) the nearest phosphates are spacefilled (cyan) (2).

We have found P22R sensitivity to non-contacted base sequence, directly affects repressor’s ability to sense sequence changes in the contacted bases. We provide evidence that this effect may be due to the spine of hydration which may run along the minor groove from one end of the binding site to the next. Together these results provide a fascinating picture of how P22R uses indirect readout in binding site discrimination.


  1. D. Watkins, Biochemistry 47, 2325-2338 (2008).
  2. L. Wu, A. Vertino, G.B. Koudelka, J.Biol.Chem 267, 9134-9135 (1992).

Lydia-Ann Harris
Gerald Koudelka

Department of Biological Sciences
University at Buffalo (SUNY)
109 Cooke Hall
Buffalo, New York, 14260