Trp repressor protein

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...

When tryptophan is lacking, a trp repressor protein (encoded by the trpR operon) is synthesized. The trp repressor dimer is inactive, cannot bind to the trp operator and so the trp operon is transcribed to produce the enzymes that then synthesize tryptophan for the cell. When tryptophan is present, tryptophan synthesis is not needed. In this situation, acting as a corepressor, tryptophan binds to the repressor and activates it so that the repressor now binds to the trp operator and stops transcription of the trp operon. 

Repression In the absence of tryptophan (Fig. la), a trp repressor protein encoded by a... 

Transcriptional control of the trp operon is mediated by the Trp repressor protein which binds to the trp operator sequence (Fig. 28.10). The DNA binding activity of the Trp repressor is controlled directly by tryptophan, which binds to the Trp repressor and functions as an effector molecule. In the presence of high concentrations... 

Fig. 5. Phosphorescence spectra at 77 K of (A) wild-type E. coli Trp repressor protein, (B) the point-mutated protein W99F, and (C) the point-mutated protein W19F. Sample excitation is at 295 nm with 16 nm bandpass emission resolution is 1.5 nm bandpass. Protein concentrations are around 0.2 mM in 0.1 M phosphate buffer, pH 7.5, containing 1 mM EDTA and 0.2 M KCl. Ethylene glycol is present at 40% (v/v). Energy transfer from Trp-19 to Trp-99 at the singlet level is responsible for the reduced contribution of Trp-19 to the phosphorescence spectrum in (A). [From M. R. Eftink, G. D. Ramsay, L. E. Bums, A. H. Maki, C. J. Mann, C. R. Matthews, and C. A. Ghiron, Biochemistry 32, 9189 (1993), with permission.]...

The Trp repressor protein is unusual in that characterization of the local environments of Trp-19 and Trp-99 based on phosphorescence and ODMR characteristics is at variance with that based on fluorescence and the X-ray crystal structure. The latter methods suggest considerably solvent exposure of both sites. In all other proteins studied to date where comparison is possible, the ODMR and phosphorescence are found to be consistent with the character of the local Trp environment as revealed by the X-ray structure. We think that the discrepancy in Trp repressor protein may be the result of a structural change induced by sample cooling that relocates Trp-99 in a buried, hydrophobic environment. 

We thank Professor Maurice Eftink for samples of native and mutated Trp repressor protein and Dr. Jose R. Casas-Finet for samples of HIV-1 nucleocapsid protein, p7. We are grateful to Dr. Laura E. Bums and Dr. Wai-Chung Lam for assistance in preparing some of the figures. Much of the work described in the final section of this chapter was partially supported by the National Institute of Environmental Health Sciences, National Institutes of Health (Grant No. ES-02662). 

J. Guenot and P. A. Kollman, Molecular dynamics studies of a DNA-binding protein—2 an evaluation of implicit and explicit solventmodels for themolecu-lar dynamics simulation of the Escherichia coli trp repressor, Protein Science, vol. 1, no. 9, pp. 1185-1205, 1992. 

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 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 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). 

Attenuation. A major mechanism of feedback repression, known as attenuation, depends not upon a repressor protein but upon control of premature termination. It was first worked out in detail by Yanofsky et al. for the trp operon of E. coli and related bacteria.184 186 Accumulation of tryptophan in the cell represses the trp biosynthetic operon by the action of accumulating tryptophanyl-tRNATlP, which specifically induces termination in the trp operon. Other specific "charged" arnino-acyl-tRNA molecules induce termination at other amino acid synthesis operons. 

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. 

Specific interactions between three different repressors and their operator binding sites. Only half the operator binding site is shown because identical contacts are made with the other half. The numbers associated with the amino acid side chains refer to the distance of amino acids from the amino-terminal end of the protein. Nucleotides are numbered from the central dyad at the operator, (a) The 434 phage repressor (b) The A repressor, (c) The trp repressor. (Source Adapted from T. Steitz, Q. Rev. Biophys. 23 236, 1990.)... 

The trp operon contains a cluster of five structural genes associated with tryptophan biosynthesis. Initiation of transcription of the trp operon is regulated by a repressor protein that functions similarly to the lac repressor. The main difference is that the trp repressor action is subject to control by the small-molecule effector, tryptophan. When tryptophan binds the repressor, the repressor binds to the trp operator. Thus, the effect of the small-molecule effector here is opposite to its effect on the lac operon. When tryptophan is present, there is no need for the enzymes that synthesize tryptophan. 

Referring to figure 30.27c, draw a detailed structure of the interaction of residue Arg69 of the trp repressor with G9 of its recognition sequence. Explain why binding of repressor proteins to DNA does not disrupt the DNA double helix. 

Crystal structures of repressors suggest a common helix-turn-helix motif for DNA binding. Repressors are protein molecules which bind to and block specific nucleotide sequences in DNA (operators), thereby regulating gene expression. Some of the repressors act without co-factors, others need a co-repressor to bind to DNA, like the tryptophan (trp) repressor, or they do not bind to the DNA operator in presence of a co-repressor like tetracyclin (TET) repressor. 

In all these crystal structures, trp repressor occurs as a dimer with intertwined subunits related by a twofold rotation axis. When tryptophan binds to the apo repressor protein, a conformational change is induced through reorientation of several hydrogen bonds (Fig. 20.19). It rotates the helix/turn/helix motif such that... 

TrpR, which is a DNA binding repressor protein, regulates transcription initiation of the E. coli trpEDCBA operon. Under tryptophan limiting conditions, TrpR represses transcription initiation, whereas repression is relieved in the presence of excess tryptophan. Once transcription initiates the elongating transcription complex is subject to control by transcription attenuation (reviewed in References 5 and 6). The leader transcript can form three RNA secondary structures that are referred to as the pause hairpin, the antiterminator structure, and an intrinsic terminator hairpin. Because the antiterminator shares nucleotides in common with the terminator, their formation is mutually exclusive. The pause hairpin has two additional roles in this transcription attenuation mechanism it serves as an anti-antiterminator stmc-ture that prevents antiterminator formation, and it codes for a leader peptide. A model of the E. coli trp operon transcription attenuation mechanism is presented in Fig. 2a. 

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