The expression of genes is regulated by a number of processes that affect the rates at which gene products are synthesized and degraded. Much of this regulation occurs at the level of the initiation of transcription and is mediated by regulatory proteins that either repress or activate transcription from specific promoters. Regulation by repressors and activators is called negative and positive regulation, respectively,
In prokaryotes, genes with interdependent functions are often clustered as a single transcriptional unit called an operon. The transcription of operon genes is generally blocked by the 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, called 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 the biological inducer, allolactose.
Regulatory proteins are DNA-binding proteins that recognize specific sequences in the DNA. Most of these proteins have distinct DNA-binding domains. Within these domains, common structural motifs involved in DNA binding are the helix-turnhelix and zinc finger motifs. Regulatory proteins also contain domains for protein-protein interactions, including leucine zipper and helix-loop-helix motifs involved in dimerization and several classes of domains involved in the activation of transcription.
The lactose operon of E. coli also exhibits positive regulation by the catabolite gene activator protein (CAP). When cAMP concentrations are high (glucose concentrations are low), CAP binds to a specific site on the DNA, stimulating transcription of the lac operon and production of lactose-metabolizing enzymes. The presence of glucose depresses cAMP concentrations, restricting expression of lac (and other) genes and suppressing the use of secondary sugars. Several operons that are coordinately regulated, as with CAP and cAMP, are referred to as a regulon.
Other mechanisms of regulation are also observed in prokaryotes. In the arabinose (ara) operon, the AraC protein acts as both activator and repressor. Some repressors, as in the ara operon and the bacteriophage λsystem, regulate their own synthesis (autoregulation). Some regulatory proteins in the ara system bind sites many base pairs distant from each other and interact by DNA looping mechanisms. Amino acid biosynthetic operons have a regulatory circuit called attenuation that uses a transcription termination site (the attenuator), modulating its formation in the mRNA by a mechanism that couples transcription and translation and responds to small changes in amino acid concentration. In the SOS system, multiple unlinked genes are repressed by a single type of repressor protein, and all of the genes are induced simultaneously when DNA damage triggers RecA protein-mediated proteolysis of the repressor. The bacteriophage λ has a complex regulatory circuit that oversees the choice between lysis and lysogeny. Two λ proteins, N and Q, act as antiterminators, modifying the host RNA polymerase so that it can bypass transcription termination sites. Finally, some prokaryotic genes are regulated by genetic recombination processes that physically move promoters relative to the genes being regulated. These diverse mechanisms permit very sensitive cellular responses to changes in environmental conditions.
Some regulation also occurs at the level of translation. The synthesis of ribosomal proteins in bacteria is mediated by a strategy in which one protein in each ribosomal protein operon acts as a ranslational repressor. The mRNA is bound by the repressor and translation is blocked only when the ribosomal protein is present in excess relative to available rRNA.
Eukaryotes employ many of the same regulatory schemes, although positive regulation appears to be more common and transcription is also accompanied by large changes in chromatin structure. Eukaryotic transcriptional activator proteins are generally required for RNA polymerase binding and activity. Some transcription factors have general functions; the TFII factors associated with RNA polymerase II, for example, are required at almost all RNA polymerase II promoters. Other transcriptional activators, unique to one gene or set of genes, have distinct domains for DNA binding and activation, and their DNA binding sites are often found hundreds of base pairs from the site where RNA synthesis begins.
Perhaps the most complex regulatory problem is the development of a multicellular animal. Here, sets of regulating genes operate in temporal and spatial succession, turning a given area of an egg cell into a predictable structure in the adult animal. Research continues into the molecular basis for this highly coordinated process.
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An excellent reference source for reuiews of many bacterial operons.
Pabo, C.O. & Sauer, R.T. (1992) Transcription factors: structural factors and principles of DNA recognition. Annu. Rev. Biochem. 61, 1053-1095.
Schleif, R. (1986) Genetics and Molecular Biology, Addison-Wesley Publishing Co., Inc., Reading, MA. Chapters 12, 13, and 14 provide ccn excellent account of the experimental basis of major concepts of gene regulation in prokaryotes.
Schleif, R. (1992) DNA looping. Annu. Reu. Biochem. 61, 199-223.
Struhl, K. (1989) Helix-turn-helix, zinc-finger, and leucine-zipper motifs for eukaryotic transcriptional regulatory proteins. Trends Biochem. Sci. 14, 137-140.
Watson, J.D., Hopkins, N.H., Roberts, J.W., Steitz, J.A., & Weiner, A.M. (1987) Molecular Biology of the Gene, 4th edn, The Benjamin/ Cummings Publishing Company, Menlo Park, CA.
Regulation of Gene Expression in Prokaryotes
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The operon model and the concept of messenger RNA were proposed in this historic paper.
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A good oueruiew of gene regulation during deuelopment.
DeRobertis, E.M., Oliver, G., & Wright, C.V.E. (1990) Homeobox genes and the vertebrate body plan. Sci. Am. 263 (July), 46-52.
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TFIID complex. J. Biol. Chem. 267, 679-682.
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Thummel, C.S. (1992) Mechanisms of transcripregulation. Sci. Am. 264 (April), 54-64. tional timing in Drosophila. Science 255, 39-40. A good description of leucine zippers.
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1. Negatiue Regulation In the lac operon, describe the probable effect on gene expression of:
(a) Mutations in the lac operator
(b) Mutations in the lacI gene
(c) Mutations in the promoter
2. Effect of mRNA and Protein Stability on Regulation An E. coli cell is growing in a solution with glucose as the sole carbon source. Tryptophan is suddenly added. The cells continue to grow, and divide every 30 min. Describe (qualitatively) how the amount of tryptophan synthase activity in the cell changes if:
(a) The trp mRNA is stable (degraded slowly over many hours).
(b) The trp mRNA is degraded rapidly, but tryptophan synthase is stable.
(c) The trp mRNA and tryptophan synthase are both degraded rapidly.
3. Functional Domains in Regulatory Proteins A biochemist replaces the DNA-binding domain of the yeast GAL4 protein with the DNA-binding domain from the λ repressor (CI) and finds that the engineered protein no longer functions as a transcriptional activator (it no longer regulates transcription of the gal operon in yeast). What might be done to the GAL4 DNA-binding site to make the engineered protein functional in activating gal operon transcription?
4. Bacteriophage λ Bacteria that become lysogenic for bacteriophage λ are immune to subsequent λ lytic infections. Why?
5. Regulation by Means of Recombination In the phase variation system of Salmonella, what would happen to the cell if the Hin recombinase became more active and promoted recombination (the switch) several times in each cell generation?
6. Transcription Attenuation In the leader region of the trp mRNA, what would be the effect of:
(a) Increasing the distance (number of bases) between the leader peptide gene and sequence 2?
(b) Increasing the distance between sequences 2 and 3?
(c) Removing sequence 4?
7. Specific DNA Binding by Regulatory Proteins A typical prokaryotic repressor protein discriminates between its specific DNA-binding site (operator) and nonspecific DNA by a factor of 105 to 106. About ten molecules of the repressor per cell are sufficient to ensure a high level of repression. Assume that a very similar repressor existed in a human cell and had a similar specificity for its binding site. How many copies of the repressor would be required per cell to elicit a level of repression similar to that seen in the prokaryotic cell? (Hint: The E. coli genome contains about 4.7 million base pairs and the human genome contains about 2.4 billion base pairs.)
8. Positiue Regulation A new RNA polymerase activity is discovered in crude extracts of cells derived from an exotic fungus. The RNA polymerase initiates transcription only from a single, highly specialized promoter. As the polymerase is purified, its activity is observed to decline. The purified enzyme is completely inactive unless crude extract is added to the reaction mixture. Suggest an explanation for these observations.
