Trp-repressor

For many years hemoglobin was the only allosteric protein whose stereochemical mechanism was understood in detail. However, more recently detailed structural information has been obtained for both the R and the T states of several enzymes as well as one genetic repressor system, the trp-repressor, described in Chapter 8. We will here examine the structural differences between the R and the T states of a key enzyme in the glycolytic pathway, phosphofructokinase. 

The trp repressor forms a helix-turn-helix motif... 

The trp repressor controls the operon for the synthesis of L-tryptophan in Escherichia coli by a simple negative feedback loop. In the absence of L-tryptophan, the repressor is inactive, the operon is switched on and the enzymes which synthesize L-tryptophan are produced. As the concentration of L-tryptophan increases, it binds to the repressor and converts it to an active form so that it can bind to the operator region and switch off the gene. 

Figure 8.18 The subunit of the trp repressor. The subunit contains 107 amino acid residues that are folded into six a helices. Helices 4 (blue) and 5 (red) form the DNA-binding helix-tum-helix motif. (Adapted from R.W. Schevitz et ah. Nature 317 782-786, 1985.)...

Figure 8.20 Schematic diagrams of docking the trp repressor to DNA in its inactive (a) and active (b) forms. When L-tryptophan, which is a corepressor, hinds to the repressor, the "heads" change their positions relative to the core to produce the active form of the repressor, which hinds to DNA. The structures of DNA and the trp repressor are outlined.

Cocrystals of Escherichia coli Trp repressor bound to an alternative operator DNA sequence. J. Mol. Biol. 234,496-498. 

The contact between protein and DNA can also be transmitted via boimd water molecules. In the crystal structure of the complex of the bacterial Trp-repressor and the cognate operator sequence are foimd only a few direct H-bonds between the amino acid residues of the protein and the bases of the recognition sequence. Rather, the contacts between protein and nucleic acid are frequently established indirectly by a chain of well-defined bound water molecules which contact the protein and the bases, and thereby function as transmitter between the protein and DNA. 

The detailed analysis of DNA structure in the region of contact with the binding protein often displays distinct divergence from the parameters of classical B-DNA structure. The specific sequence-determined conformation of the DNA is often a prerequisite for a specific recognition. This recognition mechanism is, for example, realized with the Trp-repressor, where the sequence determines a certain spatial arrangement of the... 

The strategies and mechanisms of effector molecules on regulatory DNA-binding proteins can be elucidated on the example of the Trp repressor of E. coli. 

The Trp repressor controls the transcription of a total of five enzymes required for the biosynthesis of tryptophan (Fig. 1.22a). The genes for the five enzymes are encoded in a single operon, whereby the binding site for the Trp repressor overlaps with the promoter. The bound repressor blocks the RNA polymerase s access to the promoter, thereby inhibiting transcription. 

The enzymes of Trp-biosynthesis are only required if too little tryptophan is available to the bacteria from the growth medium. In such a case the Trp requirement is fulfilled by the cell s own Trp biosynthesis. If however, there is enough Trp supplied by the medium, then it is prudent to shut down the Trp operon. The sensor is the Trp concentration. The Trp repressor registers the current Trp concentration with the help of its own Trp binding site. If a great deal of Trp is present, then the Trp binding site of the repressor is occupied by Trp. The Trp repressor binds Trp with high affinity (Kd=10 -10 M), upon which transcription of the operon is then blocked. 

At low Trp concentration the Trp repressor is mainly in the unbound, inactive form. The free form of the Trp repressor binds with a ca. lO -fold lower affinity to the recognition sequence than the Trp-bound form. The promoter remains free under these conditions and transcription of the genes for Trp biosynthesis can occur. The shutting on and off of the Trp operon is based on the disparate DNA affinities of the free and Trp-boimd repressor. ... 

The comparison of the structure of a binding protein in the inactive form and in the active form boimd to DNA gives an impression of the conformational changes correlated with binding of effector molecules. The Trp repressor is, next to the Lac repressor from E. coli, one of the few examples in which the structural basis for the difference in DNA-binding affinity of the inactive vs. active form is understood (Fig. 1.22b). 

FIGURE 28-20 Trp repressor. The repressor is a dimer, with both subunits (gray and light blue) binding the DNA at helix-turn-helix motifs (PDB ID 1TRO). Bound molecules of tryptophan are in red. 

Figure 5-35 Stereoscopic drawings illustrating the binding of a dimeric molecule of the Trp repressor protein to a palindromic sequence in DNA. (A) Schematic view showing structures of the aporepressor (partly shaded gray) and the holorepressor with bound tryptophan (unshaded) are superimposed. Cylinders represent the a helices in (B). From Zhang et al.wi (B) MolScript ribbon diagram with a few side chains that interact with the DNA shown. Two tandemly bound dimeric repressor molecules are shown. Two bound molecules of tryptophan are visible in each dimer. The DNA is drawn as a double helix with lines representing the base pairs. From Lawson and Carey.405...

Examination of DNA-regulatory protein complexes have permitted reasonable guesses to be made about the precise nature of the contacts between amino acid side chains and DNA in many cases. Figure 30.27 illustrates three examples One for the 434 phage repressor (fig. 30.27a), one for the A cl repressor (fig. 30.27b), and one for the trp repressor (fig. 30.27c). In all cases only half-sites are depicted because symmetry considerations dictate that the two halfsites should have virtually identical structures. 

Despite the apparent advantages in the amide-containing amino acids, we do not always find amides used as part of the recognition sequence. For example, the trp repressor does not use any amides. The basic amino acids, lysine and arginine, and the hydroxylic amino acid, threonine, are the main interacting amino acids in the trp repressor. The side... 

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