Repressor proteins, operons

Figure 39-2. The positional relationships of the structural and regulatory genes of the lac operon. lacZ encodes 3-galactosidase,/ocT encodes a permease, and lacA encodes a thiogalactoside transacetylase. lad encodes the lac operon repressor protein.

Emulation by activator and repressor proteins in the lactose operon... 

In addition, the i gene, which encodes the lac repressor protein, is also considered part of the operon although it is located at a distant site in the DNA. The i gene is constitutively expressed (not regulated) thtis, copies of the lac repressor protein are always in the cell. 

In bacteria, genes that encode products with interdependent functions are often clustered in an operon, a single transcriptional unit. Transcription of the genes is generally blocked by binding of a specific repressor protein at a DNA site called an operator. Dissociation of the repressor from the operator is mediated by a specific small molecule, an inducer. These principles were first elucidated in studies of the lactose (lac) operon. The Lac repressor dissociates from the lac operator when the repressor binds to its inducer, allolactose. 

The r-protein operons are regulated primarily through a translational feedback mechanism. One r-protein encoded by each operon also functions as a translational repressor, which binds to the mRNA... 

The lac operon is ordinarily subject to repression and is activated by the presence of an inducer, now known to be allolactose, D-Galp-(il— 6-d-G1c. However, in experimental work artificial inducers such as isopropyl-[5-o-thiogaIactoside (IPTG) are most often used. Jacob and Monod postulated that the free repressor protein binds to the operator. In the presence of the inducer a conformational change takes place, destroying the affinity of the repressor protein for the operator site. Thus, in the presence of inducer the operator is not blocked, and the transcription takes place. Such an operon is said to be negatively controlled and inducible. 

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. 

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. 

The lacl gene has its own promoter (Pkd) to which RNA polymerase binds and initiates transcription. In the absence of an inducer, the lacl gene is transcribed, producing lac repressor mRNA and hence lac repressor protein monomers. These monomers assemble to form active tetramers which bind to the lac operator site, 0, , and prevent transcription of the lac operon. In the presence of an inducer (such as allolactose or IPTG), the inducer binds to the repressor and changes its conformation, reducing its affinity for the lac operator. Thus the repressor now dissociates and allows RNA polymerase to transcribe the lac operon. 

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. 

In end-product repression (see later), it was postulated that the repressor protein is inactive until it binds the repressing metabolite (corepressor) to form an active repressor-corepressor complex that can combine with the operator gene to block synthesis at the operon. 

The regulation of bacterial transcription is well illustrated by the lactose operon (lac operon) of the colon bacterium Escherichia coli in which the upstream region successively (from the 5 end of the sense strand) includes a promoter (P ) for the gene (I) coding for a repressor protein (the lac repressor), a CRP binding site , the promoter for the lac operon (P), and finally an operator site (O) that prefaces the Z, Y and A structural genes of the operon ... 

Fig. 6. Occurrence of the CXCX(4 5) CXC consensus motif. CopY, cop operon repressor protein from Enterococcus hirae Mad, transcription factor for the Ctrl copper transporter of Saccharomyces cerevisiae AMTl, transcription factor for metal-lothionein from Candida albicans ACEl, transcription factor for metallothionein from Sa. cerevisiae Grisea, MACl orthologue of Podospora anserina MT-2 p-domain, N-terminal domain of human metallothionein-2.

A second demonstration of participation in renaturation of heat-inactivated proteins by dnaK is provided by experiments with a temperature-sensitive repressor protein of bacteriophage X, XcI857 protein (Gaitanaris et ai, 1990). Activities of the Xcl wild-type and mutant proteins were measured in an in vitro operator DNA binding assay and by in vivo expression from a Xcl-regulated operon fusion of XPrOr and the... 

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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