Structure of Cro and cI Repressors

Cro Structure - Cro is a homodimer of 66-residue subunits folded into three -helical regions and three strands (Figure 26.28). Two of the helices, numbered 2 and 3 in the figure, are separated by a short turn, forming a helix-turn-helix motif (see here and here). In the Cro dimer the two number 3 helices are 3.4 nm apart, the length of one turn of the DNA double helix. This distance is consistent with the two subunits binding on the same side of the DNA helix, in adjacent major groove sites, with the number 3 helices lying lengthwise in the grooves.

The amino acid sequence within and between helices 2 and 3 show remarkable homology with corresponding sequences in a large family of sequence-specific DNA-binding proteins but not with DNA-binding proteins that showed no sequence preference. The helix-turn-helix motif is a commonly evolved structural element in transcriptional regulatory proteins, at least in prokaryotes. A different motif, the zinc finger, is present in a large number of eukaryotic DNA-binding proteins (and a few known prokaryotic proteins). Other structural patterns of DNA-binding proteins, including the helix-loop-helix and the leucine zipper, have been described. These motifs are discussed here.

cI Structure - The cI repressor also has a helix-turn-helix motif. The relevant three-dimensional similarities for Cro, cAMP receptor protein (CRP, see here), and cI repressor are shown in Figure 26.29. cI repressor contains an additional binding determinant-a pair of "arms," or short polypeptide segments that extend from helix 1. They can be seen in Figure 26.1, extending around the helix and establishing contacts on the other side of the DNA duplex. These arms probably explain why cI binds more tightly to its operators than Cro does.

The -3 helix of cI is called the recognition helix, because when cI is bound to DNA, its position deep within the major groove allows it to contact specific DNA bases and hence to determine sequence specificity of binding. The -2 helix is in contact primarily with DNA phosphates. These electrostatic contacts strengthen binding but do not contribute to specificity.

Amino acids and DNA binding - Amino acid sequence homologies among the DNA binding regions for Cro and cI are shown in Figure 26.30. The sequences, though similar, are not identical, which explains how Cro and cI repressors differ in their relative affinities for different operators. The residues common to both proteins are in contact with DNA sequence elements common to all of the operators (Figure 26.31). In both proteins a glutamine residue interacts with one A-T base pair, as shown. cI repressor, which binds most strongly to O R1, establishes specificity through a contact in O R 1 with a unique alanine residue, whereas Cro, which binds most strongly to O R 3, can be in contact with three specific base pairs in O R3 with unique asparagine and lysine residues.

See also: cAMP receptor protein (CRP), trp Operon Regulation

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> 1. Helix-Loop-Helix Binding Domains

> 2. Helix-Loop-Helix Proteins