Repressor lambda

The fundamental unit of tertiary structure is the domain. A domain is defined as a polypeptide chain or a part of a polypeptide chain that can fold independently into a stable tertiary structure. Domains are also units of function. Often, the different domains of a protein are associated with different functions. For example, in the lambda repressor protein, discussed in Chapter 8, one domain at the N-terminus of the polypeptide chain binds DNA, while a second domain at the C-terminus contains a site necessary for the dimerization of two polypeptide chains to form the dimeric repressor molecule. 

The x-ray structure of the DNA-binding domain of the lambda repressor is known... 

Superficially, the lambda repressor protein is very different from lambda Cro. The polypeptide chain is much larger, 236 amino acids, and is composed of two domains that can be released as separate fragments by mild proteolysis. In repressor the domain responsible for dimerization is separate from the... 

Figure 8.6 The N-terminal domain of lambda repressor, which binds DNA, contains 92 amino acid residues folded into five a helices. Two of these, a2 (blue) and a3 (red) form a helix-turn-hellx motif with a very similar structure to that of lambda Cro shown In Figure 8.4. The complete repressor monomer contains in addition a larger C-termlnal domain. (Adapted from C. Pabo and M. Lewis, Nature 298 443-447, 1982.)...

The x-ray structure of the N-terminal DNA-binding domain of the lambda repressor was determined to 3.2 A resolution in 1982 by Carl Pabo at Harvard University and revealed a structure with striking similarities to that of Cro, although the p strands in Cro are replaced by a helices in repressor. 

In spite of the absence of the C-terminal domains, the DNA-binding domains of lambda repressor form dimers in the crystals, as a result of interactions between the C-terminal helix number 5 of the two subunits that are somewhat analogous to the interactions of the C-terminal p strand 3 in the Cro protein (Figure 8.7). The two helices pack against each other in the normal way with an inclination of 20° between the helical axes. The structure of the C-terminal domain, which is responsible for the main subunit interactions in the intact repressor, remains unknown. 

This model of Cro binding to DNA was arrived at by intuition and clever model building. Its validity was considerably strengthened when the same features were subsequently found in the DNA-binding domains of the lambda-repressor molecule. The helix-turn-helix motif with a recognition helix is present in the repressor, and moreover the repressor DNA-binding domains dimerize in the crystals in such a way that the recognition helices are separated by 34 A as in Cro. 

Figure 8.11 The DNA-binding domain of 434 repressor. It is a dimer in its complexes with DNA fragments. Each subunit (green and brown) folds into a bundle of four a helices (1-4) that have a structure similar to the corresponding region of the lambda repressor (see Figure 8.7) including the helix-turn-helix motif (blue and red). A fifth a helix (5) is involved in the subunit interactions, details of which are different from those of the lambda repressor fragment. The structure of the 434 Cro dimer is very similar to the 434 repressor shown here.

The three-dimensional strucmres of Cro and of the lambda repressor protein have been determined by x-ray crystallography, and models for their binding and effecting the above-described molecular and genetic events have been proposed and tested. Both bind to DNA using hehx-turn-helix DNA binding domain motifs (see below). 

Detailed analysis of the lambda repressor led to the important concept that transcription regulatory proteins have several functional domains. For example, lambda repressor binds to DNA with high affinity. Repressor monomers form dimers, dimers interact with each other, and repressor interacts with RNA polymerase. The protein-DNA interface and the three protein-protein interfaces all involve separate and distinct domains of the repressor molecule. As will be noted below (see Figure 39—17), this is a characteristic shared by most (perhaps all) molecules that regulate transcription. 

Once the lambda repressor has been inactivated, the positive and negative control exerted by this repressor are abolished, and new transcriptional events can be initiated. The most important transcriptional event is that involved in the synthesis of another lambda protein called Cro. coded by a gene called cro. The gene cro is located almost adjacent to the gene cl which codes for lambda repressor. 

In the region separating these two genes are two kinds of sites, promoters and operators, to which each of the proteins of the switch can bind. When lambda repressor is bound to its operator, it covers... 

Bashford D Weaver D. L. and Karplus M. Diffusion-collision model for the folding kinetics of the lambda-repressor operatorbinding domain. J. Biomol. Str. Dyn. (1984) 1 1243-1255. 

Nelson, H. C. and Sauer, R. T. (1986). Interaction of mutant lambda repressors with operator and non-operator DNA. /. Mol. Biol. 192,27-38. 

Deb, S., Bandyopadhyay, S., Roy, S. (2000). DNA sequence dependent and independent conformational changes in multipartite operator recognition by lambda-repressor. Biochemistry, 39(12), 3377-3383. 

Figure 28-11 Genetic and physical map of the X phage genome. After Szybalski. See Honigman et al.255 for a more detailed diagram of the immunity region. The gene for the lambda repressor is labeled C[.

The fastest-folding small proteins generally fold on much slower time scales than the time scale of formation of secondary structure. The speed record is currently held by lambda(6-85), a truncated, monomeric form of the N-terminal domain of lambda repressor, which refolds with a half-life of approximately 140 fjis. A thermostable lambda(6-85) variant with alanine substituted for glycine residues 46 and 48 in the third helix folds faster in dilute solutions of de-naturant, with an extrapolated half-life of less than 10 /us in water.13 Cold-shock protein CspB from Bacillus subtilis folds in about 1 ms.61 Engineered mutants of the P22 Arc repressor62 and CI263 fold in a fraction of a millisecond. 

Gronenbom, B. (1976) Overproduction of the phage lambda repressor under control of the lac promoter of Escherichia coh Mol. Gen. Genet 148,243—250. 

Hu,J. C., O Shea, E. K., Kim, P. S., and Sauer, R. T. (1990). Sequence requirements for coiled-coils - analysis with lambda-repressor-Gcn4 leucine zipper fusions. Science 250, 1400-1403. 

Breiner KM, Daugherty MA, Oas TG, Thorp HH. An anionic diplatinum DNA photocleavage agent chemical mechanism and footprinting of lambda repressor. I Am Chem Soc 1995 117 11673. 

Fig. 9. Two hydrogen bonding motifs found in the lambda repressor-operator complex. Taken from Ref. 50...

Jayaram B, DiCapua FM, Beveridge DL (1991) A theoretical study of polyelectrolyte effects in protein-DNA interactions Monte Carlo free energy simulations on the ion atmosphere contribution to the thermodynamics of lambda repressor-operator complex formation. J Am Chem Soc 113 5211-5221... 

Gaitanaris, G. A., Papavassiliou, A. G., Rubock, P., Silverstein, S.., and Gottesman, M. E. (1990). Renaturation of denatured lambda repressor requires heat shock proteins. Cell (Cambridge, Mass.) 61, 1013-1020. 

Samiee, K. T., Foquet, M., Guo, L., Cox, E. C., and Craighead, H. G. (2005). Lambda repressor oligomerization kinetics at high concentrations using fluorescence correlation spectroscopy in zero-mode waveguides. Biophys. J. 88 2145-2153. 

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