Functional repressor

A repressor molecule can bind to an operator and block transcription a corepressor is a compound which binds to a protein to give a functional repressor, which can then bind to an operator. 

Site-directed mutagenesis of DtxR has indicated that, of the two metal-ion binding sites identified earlier, only one is essential for repressor activity [22, 27, 31]. Individual mutation of Met 10, Cys 102, Glu 105 or His 106, the primary metal-ion binding ligands, to Ala resulted in non-fimctional repressors [22, 31]. Individual mutation of residues His 79, Glu 83 or His 98, the ligands to the ancillary site, to Ala resulted in still functional repressors [22]. The implication of these studies is that one of the metal-ion binding sites is essential to repressor activity, while the other is not, or has at best a modulatory function. 

The Jacob-Monod theory predicts (or, more correctly, was based on) two kinds of control-deficient mutations. Mutations in the regulatory gene prevent formation of active repressor. Lack of a functional repressor leads to uncontrolled high-level expression of the structural... 

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 434 Cro molecule contains 71 amino acid residues that show 48% sequence identity to the 69 residues that form the N-terminal DNA-binding domain of 434 repressor. It is not surprising, therefore, that their three-dimensional structures are very similar (Figure 8.11). The main difference lies in two extra amino acids at the N-terminus of the Cro molecule. These are not involved in the function of Cro. By choosing the 434 Cro and repressor molecules for his studies, Harrison eliminated the possibility that any gross structural difference of these two molecules can account for their different DNA-binding properties. 

More than 30 years ago Jacob and Monod introduced the Escherichia coli lac operon as a model for gene regulation. The lac repressor molecule functions as a switch, regulated by inducer molecules, which controls the synthesis of enzymes necessary for E. coli to use lactose as an energy source. In the absence of lactose the repressor binds tightly to the operator DNA preventing the synthesis of these enzymes. Conversely when lactose is present, the repressor dissociates from the operator, allowing transcription of the operon. 

The lac repressor monomer, a chain of 360 amino acids, associates into a functionally active homotetramer. It is the classic member of a large family of bacterial repressors with homologous amino acid sequences. PurR, which functions as the master regulator of purine biosynthesis, is another member of this family. In contrast to the lac repressor, the functional state of PurR is a dimer. The crystal structures of these two members of the Lac I family, in their complexes with DNA fragments, are known. The structure of the tetrameric lac repressor-DNA complex was determined by the group of Mitchell Lewis, University of Pennsylvania, Philadelphia, and the dimeric PurR-DNA complex by the group of Richard Brennan, Oregon Health Sciences University, Portland. 

Selection of these regulatory mutants is often done by using toxic analogues of amino adds for example p-fluoro-DL-phenylalanine is an analogue of phenylalanine. Mutants that have no feedback inhibition or repression to the amino add are also resistant to the analogue amino add. They are therefore selected for and can be used to overproduce the amino add. Some amino add analogues function as false co-repressors, false feedback inhibitors or inhibit the incorporation of foe amino acid into foe protein. 

The human PR exists as two functionally distinct isoforms PRA and PRB transcribed from two promoters from a single gene. PRA lacks the N-terminal 164 aa and is a 769 aa protein. PRB functions as a transcriptional activator in most cell and promoter contexts. In contrast, PRA is transcriptionally inactive and functions as a strong ligand-dependent transdominant repressor of SHR transcriptional activity. Different cofactor interactions were demonstrated for PRA and PRB, probably due to an inhibitory domain within the first 140 aa of PRA, which is masked in PRB. Both PR isoforms however, repress estradiol-induced ER activity when liganded. Several other mRNA isoforms are present in PR-positive tissues such as breast cancer with unknown clinical significance. 

Cronin CA, Gluba W, Scrable H (2001) The lac operator-repressor system is functional in the mouse. Genes Dev 15 1506-1517... 

Malim MH, Bohnlein S, Hauber J, CuUen BR (1989) Functional dissection of the HlV-1 Rev transactivator-derivation of a trans-dominant repressor of Rev function. Cell 58 205-214... 

A third class of sequence elements can either increase or decrease the rate of transcription initiation of eukaryotic genes. These elements are called either enhancers or repressors (or silencers), depending on which effect they have. They have been found in a variety of locations both upstream and downstream of the transcription start site and even within the transcribed portions of some genes. In contrast to proximal and upstream promoter elements, enhancers and silencers can exert their effects when located hundreds or even thousands of bases away from transcription units located on the same chromosome. Surprisingly, enhancers and silencers can function in an orientation-independent fashion. Literally hundreds of these elements have been described. In some cases, the sequence requirements for binding are rigidly constrained in others, considerable sequence variation is... 

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. 

As part of a study of the functionalities of inducers wich are necessary for recognizing the repressor protein we have previously shownl that ... 

---