MCB 201 Gene Expression - Spring Semester 2003
Lecture 12 (Regulation of Transcription Initiation)
1. Introduction: An underlying principle of molecular cell biology is that the structure and function of cells are determined by the particular kinds and amounts of proteins they contain. We have learned that gene expression is the process by which genetic information encoded in genes is transmitted within cells and decoded by the processes of transcription and translation, respectively, into proteins. It follows then that it is differential gene expression that creates the different protein inventories of different cells. Differential gene expression is accomplished by regulation of gene expression. There are many different mechanisms of regulation of gene expression that have been discovered, but most frequently, regulation occurs at the very first step in the process, which is initiation of transcription. The classic experiments in regulation of transcription were carried by Francois Jacob and Jacques Monod in Paris in the 1950's. Although the details of regulation of transcription in prokaryotic and eukaryotic cells are quite different, there are some general principles from the work by Jacob and Monod which carry over into animal cells:
2. What is the purpose of gene regulation. In bacteria, it is used to allow bacteria to respond to their environment, especially the nutritional composition of the environment, but also to environmental stressors. Thus the main focus is on inducible proteins that allow bacteria to use particular nutrients or that help bacteria to mount defensive responses. These proteins allow cells to adjust growth and division to environmental conditions. In eukaryotic cells, gene regulation is most often associated with embryonic development and cell differentiation. However, eukaryotic cells can also respond to protein-based inducible defenses, such as the heat shock response.
3. Figure 10-1. The lac operon includes three genes: lacZ, which encodes beta-galactosidase; lacY, which encodes lactose permease; and lacA, which encodes thiogalactoside transacetylase. The lac I gene plus the transcriptional control region shown in yellow to the left of the lacZ gene constitute the regulatory region. The region shown in yellow contains DNA sequence that are binding sites for RNA polymerase (promoter region) and lac repressor (operator region).
4. Figure 10-2. Jacob and Monod model of transcriptional regulation of the lac operon by lac repressor. The key to regulation by the repressor is that in the absence of lactose in the environment of E coli, the proteins encoded by the lactose operon, which take up lactose into cells and break it down to glucose and galactose for use in energy metabolism, are not needed by the cell, so the repressor proteins are made and they bind to the operator region of the DNA, blocking the binding of RNA polymerase. When lactose is present in the environment, some of it is brought into cells by a low level of lactose permease that is always present (the repressor is not completely effective in keeping the lac operon turned off). The lactose is modified slightly to allolactose which can bind to repressor protein. The repressor with bound allolactose has a different shape or conformation than unbound repressor and cannot bind to the operator region. This allows RNA polymerase to bind to the adjacent and partially overlapping promoter site and to initiate transcription on the lactose operon.
5. Figure 10-3. Experimental demonstration that operator constitutive mutations are cis-acting. Cis-acting mutations define binding sequences on DNA for regulatory proteins. The strategy behind this experiment is to use two copies of the lactose operon, one which is a chromosomal copy carrying the operator constitutive mutation and a mutated lacZ gene such that no beta-galactosidase can be produced from this copy of the gene. Then, a second copy is introduced into the same cell on an F-plasmid, this time a completely normal and functional copy of the lactose operon. The hypothesis to be tested is that the operator constitutive mutation only affects the genes under its control on the same DNA segment and it does not activate the copy of the lac Z gene encoding beta-galactosidase on the plasmid. We call this a cis-acting mutation. If the hypothesis is false, the operator constitutive mutation will somehow be able to act in trans to turn on production of beta-galactosidase, which we can measure in the cell lysate. IPTG is a nonhydrolyzable analog of lactose that is used in the lab to turn on the lactose operon. No IPTG inducer is used in this experiment, since it would inhibit the lac repressor and cause activation of the lac Z gene on the plasmid, thus masking any potential effect of the operator constitutive mutation on the plasmid copy of the operon. But the result is that there is no effect, and the operator constitutive mutation is indeed a cis-acting mutation.
6. Figure 10-4. Experimental demonstration that the lacI+ gene is trans-acting. Trans-acting mutations define genes that encode regulatory proteins. The experimental strategy again involves the use of two copies of the lactose operon, one on the bacterial chromosome and one on an F-plasmid. In this case, the chromosomal copy has a mutated lacI gene which encodes a defective lac repressor. Without a functional repressor, the lactose operon is transcribed and beta-galactosidase is produced. The job of the lactose operon on the F-plasmid is to provide a functional copy of the lacI gene. The hypothesis that we are testing is that this repressor can bind to the operator site on the F-plasmid, and it can also diffuse over to the operator site on the chromosomal copy of the operon and inhibit its expression, i.e. the lacI gene is trans-acting, in 'genetic-speak'. Again, no IPTG is used in this protocol, since it would bind to lactose repressor leading to activation of the operons, thus masking any trans effect.
7. Figure 7-17. Membrane-hybridization assay for detecting nucleic acids. This assay will be used in the next experiment that I will describe to demonstrate that activation of the lac operon leads to the accumulation in cells of RNA transcribed from the lac operon. This is a flexible assay since a variety of double stranded nucleic acids will stick to the membrane filter including DNA-DNA, RNA-RNA and DNA-RNA hybrids.
8. Figure 10-5. Biochemical demonstration that inducer leads to an increase in lac operon transcription. The purpose of this experiment is to detect newly made mRNA from the lactose operon after bacterial cells are exposed to an inducer like IPTG. The operon is induced in cells that have taken up radioactive uridine. The uridine is incorporated into all RNA that is being made from the lactose operon and any other genes that are being transcribed. The trick then is to sort out the lactose operon RNA from the others. To do this, cell lysates are incubed under DNA hybridization conditions on a membrane to which single-stranded DNAs containing the lactose operon genes are attached. The nonhybridizable radioactive RNA from other genes is washed away and the filter is counted to determine how much radioactive RNA encoding the lactose operon has been made at each time after induction.