II. Cro Structure
III. Cro-Operator DNA Interaction
Note: This exhibit is best viewed if the cue buttons () are pressed in sequence and if the viewer does not independently manipulate the molecule on the left.
Cro and repressor compete for control of an operator region containing three operators that determine the state of the lytic/lysogenic genetic switch. If this competition is won by repressor, transcription of the cro gene is blocked and repressor synthesis is maintained. Lysogeny will result. A competition won by cro, however, means that the late genes of phage l will be expressed; this will result in lysis. In this case, cro blocks transcription that occurs from PRM, the promoter that is responsible for the maintenance of repressor transcription. If you are unfamiliar with the mechanisms of the lambda genetic switch, you may wish to study Figure 1 (Genetic Switch Overview) and Figure 2a,b (Operator DNA Sequences) before moving on to structural stuidies of cro. Reload molecule for the next section.
Cro (and repressor) contains a helix-turn-helix (HTH) motif comprising three a helices, as shown at left for a phage l cro monomer bound to a consensus operator half site . Two of these (a2 and a3) are separated by a short turn and are held at 550 with respect to one another. a3, the recognition helix, fits into the major groove operator DNA.
In the l cro dimer the recognition helices (a3) of each monomer are ~ 34 Å apart, thus positioned to bind in the major grooves of each operator half site, separated by ~10 base pairs (1 turn of B-form DNA). The primary interface between monomers is a series of hydrogen bonds between beta strands 3 (b3) of each monomer, extending a 3-stranded sheet in each monomer to a 6-stranded, antiparallel beta sheet in the dimer . Two points of hydrophobic contact also contribute to dimer stability: 1) phe58 of one monomer packs against ile40 in the core of the other ; 2) hydrophobic methionines at the amino terminus of each monomer are juxtaposed .
As shown for phage 434 cro - OR1 interaction at left , the cro dimer binds the two operator half sites, with the recognition helix of each monomer (a3) inserted into the DNA's major groove. Diverse molecular interactions between cro and operator provide cro with the ability to bind to generic operator DNA, as well as the ability to discriminate between operators.
Generic Operator Recognition
The a3 recognition helices are oriented with their axes parallel to the major groove. The side chains of each helix are thus positioned to interact with the edges of base pairs on the floor of the groove . Both cro and repressor of phage 434 contain glutamines (28 and 29) in a3 that bond with major groove bases. For example, gln28 and gln29 of one cro monomer form H-bonds with bases of the first two base pairs of an OR1 half site . [You may wish to consult the repressor exhibit to compare repressor's gln28 and gln29 bonding to that of cro]. Not surprisingly, these residues are essential for both cro and repressor function: mutation of them renders phage inviable.
Non-specific interactions also help anchor cro and repressor to operator DNA. These include H-bonding between main chain NH groups and phosphate oxygens of the DNA in the region of the operator, shown at left for 434 cro and OR1 .
Specific Operator Recognition
As discussed previously, the differential affinity of cro and repressor dimers for the OR1, OR, and OR3 is the mechanistic crux of the temperate phage genetic switch. What is the structural basis for the discrimination between operators with quite similar sequences?
It is interesting that this differential affinity is not the result of sequence specific binding of base pairs by amino acid side chains of the recognition helix. Rather, it is a consequence of protein-induced conformational changes in the DNA. The ability of operator DNA to adapt a shape that optimizes surface complementarity with the protein determines the affinity of either cro or repressor for a particular operator.
Comparing phage 434 OR1 operator DNA structures (cro-bound and unbound) shows that cro distorts normal B-form DNA conformation. OR1 DNA is bent (curved) by cro, and the middle region of the operator is overwound, reflected in a reduced distance between backbones in the minor groove. See methods for a description of the model shown.
The cro protein also changes conformation upon DNA binding. The beta strands at the monomer interface (see above) twist, rotating the monomers 40o with respect to each other. This can be seen at left by comparing the backbone conformations of a phage l cro dimer bound to operator and free l cro dimer (see methods).
Returning to a view of 434 cro - OR1 interaction, considerable protein-DNA complementarity is evident. OR3 likely adapts a shape that provides for even more interaction with cro (not shown), accounting for cro's greater affinity for OR3. Please consult the repressor exhibit for further treatment of protein-operator interactions.
Albright, RA and Matthews, BW (1998) Crystal Structure Of Lambda-Cro Bound To A Consensus Operator At 3.0 Angstrom Resolution J.Mol.Biol. 280: 137.
Albright, RA and Matthews, BW (1998). How Cro and l-repressor Distinguish Between Operators: The Structural Basis Underlying a Genetic Switch. PNAS 95: 3431-3436.
Brennan, RG, Roderick, SL, Takeda, Y, and Matthews, BW (1990). Protein-DNA Conformational Changes in the Crystal Structure of a l Cro-operator Complex. PNAS 87: 8165-8169.
Mondragon, A, and Harrison, SC (1991). The Phage 434 Cro/OR1 Complex At 2.5 Angstroms Resolution. J.Mol.Biol. 219: 321.
Ohlendorf, DH, Tronrud,
DE, Matthews, BW (1998). Refined Structure of Cro Repressor Protein from Bacteriophage
Lambda Suggests Both Flexibility and Plasticity. J. Mol. Biol. 280: 129-136.