Textbook assignment: Chapter 8, Section 8.3, Pages 229-233.
Section 8.4, pages 233 - 237 is optional -- see the appendix to these notes.
Major concepts:
The tryptophan operon: The trp operon contains the structural genes for five enzymes that are required for the biosynthesis of tryptophan. These enzymes are needed only when tryptophan is not available from the extracellular environment. Coordinated transcription of these five genes is controlled in two very different ways: 1) through the use of an end-product activated repressor protein; and 2) through the use of a novel leader-attenuator mechanism. The operon itself consists of a promoter, an operator site, the leader-attenuation site (described later), and the five structural genes (figure 8.14). The operon (trpFDCBA) is located at 28 minutes on the E coli genetic map (figure 7.22) and is oriented in the counterclockwise direction. The trp repressor protein is coded from a separate gene, trpR, located at 99.7 minutes on the map and oriented in a clockwise direction.
End product-mediated repression of the trp operon: Repression of the trp operon utilizes an operator site and a repressor protein whose overall functions resemble those of the lac operator and repressor. However, by itself, the trp repressor protein is inactive and is therefore referred to as an aporepressor. For repression to occur, tryptophan must be present at a sufficiently high concentration to bind to the aporepressor protein and convert it to an active repressor that will bind to the operator and stop transcription (figure 8.15). Thus, tryptophan serves as a corepressor. When the environment contains enough tryptophan, the operon is repressed, allowing the cell to save the energy that would otherwise be expended for the transcription and translation of the five gene products coded for in the operon. .
Derepression: When the level of available tryptophan drops, mass action causes it to dissociate from the repressor protein, which then becomes non-functional. This causes derepression to occur, giving the cell the capability of synthesizing its own tryptophan. Thus absence of the corepressor (tryptophan) inactivates a negative control system, rather than directly stimulating transcription. This arrangement makes it relatively easy for the cells to respond quickly to the absence of tryptophan by derepressing expression of genes whose function is needed only for the synthesis of tryptophan. The lac operon is also activated by derepression, but the mechanism is exactly the opposite from that for the trp operon. The lac repressor protein alone is active, but is deactivated by allolactose, whereas the trp repressor protein alone is inactive, but is activated by tryptophan.
Attenuation of transcription in the trp operon: The attenuation mechanism is described in detail in the textbook. It provides a second level of control that allows the extent of transcription of the trp operon to be modified in response to the amount of charged trp- tRNA in the cell. This control system, which is only operational when transcription of the operon has been derepressed, provides a more sensitive index of whether there is enough tryptophan present to support protein synthesis. Under conditions where protein synthesis is not being inhibited by an insufficient supply of charged trp-tRNA, transcription of the operon is prematurely terminated before reaching the first structural gene in the operon (trpE).
Control over termination of transcription: In brief summary, the first part of the trp transcript includes four regions (1-4) that can either form two stem loops by pairing 1:2 and 3:4, or a single stem loop by pairing 2:3, with regions 1 and 4 unpaired (figure 8.17). (Note that our textbook numbers the loops, rather than the regions that form them. The 1:2 stem loop is referred to as hairpin #1, the 2:3 stem loop is hairpin #2, and the 3:4 stem loop is hairpin #4). Immediately following region 4, there is a sequence of eight uracils (U). If the 3:4 stem loop (hairpin #3) is allowed to form as regions 3 and 4 are being transcribed, that loop together with the oligo-U sequence that is transcribed immediately afterward generate a prokaryotic transcription termination signal (figure 8.16). (At this point, you may want to go back to page 70 and figure 3.13 for a review of the termination of prokaryotic transcription). Formation of the stem-loop termination signal causes transcription to stop before it reaches the structural genes for the five enzymes coded by the trp operon, which begin just downstream from the oligo-U sequence. The leader-attenuator system operates by controlling the extent of formation of the 3:4 loop, and thus of the termination signal, as described below.
Ribosomal stalling due to insufficient charged tryptophane-tRNA: There is an open reading frame for a short peptide of 14 amino acids, with its translational initiation site just upstream from region 1 (figure 8.17a). The coding sequence includes two trp codons side by side in positions 8 and 9, located at the start of region 1. Translation starts soon after the free 5'-end of the mRNA leaves the RNA polymerase. If tryptophan levels inside the cell are low, charged trp-tRNA will be in short supply and the ribosome will temporarily stall at the two trp codons in region 1. This will prevent region 1 from forming a stem loop with region 2 (hairpin #1), which will encourage regions 2 and 3 to form a stem loop (hairpin #2). This in turn keeps regions 3 and 4 from forming the stem-loop (hairpin #3) plus oligo-U termination signal. In the absence of the termination signal, the RNA polymerase continues beyond the attenuator site and begins transcription of the structural genes (figures 8.17c and 8.18a).
Attenuation when adequate trp-tRNA is present: On the other hand, if there is enough intracellular trp-tRNA so the ribosome can move quickly past the two trp codons, the remainder of the short (14 amino acid) leader peptide is synthesized and the ribosome moves into a position that blocks region 2 from base pairing with region 3 (and ultimately departs form the transcript as translation of the peptide is terminated). This leaves region 3 available to pair with region 4 and thus to form the stem-loop termination signal before the RNA polymerase has moved beyond the end of the poly (U) sequence. The net result is early termination of transcription before any of the structural genes are transcribed (figure 8.18b). In addition, the relative probabilities of continuing transcription vs. terminating it are highly responsive to small variations in the intracellular concentration of charged trp-tRNA, providing a much more sensitive control system than can be achieved by interaction of free tryptophan with the trp repressor protein as the only control mechanism.
Other attenuation systems: A similar combination of end product-mediated repression and leader attenuation is also used for operons encoding enzymes for synthesis of a number of other amino acids in various bacteria. In a more extreme case, an operon encoding nine enzymes for the biosynthesis of histidine in Salmonella typhimurium is controlled entirely by a leader-attenuation mechanism, with no repressor protein at all. For additional information on attenuation systems go to the textbook publisher's web page, click on the chapter by chapter resources section on the left. When the new page appears, select chapter 8, then select hypercontents from the choice of resources and click on "Go". You will be offered a menu that includes a section on the mechanisms of attenuation, as well as other interesting topics related to chapter 8 of the textbook. .
APPENDIX: BACTERIOPHAGE LAMBDA
Bacteriophage lambda: This is not be required material for the Fall 2000 class. However, I would encourage you to read through the unusual mechanisms that are employed by bacteriophage lambda in determining whether an infection will be lytic or lysogenic. These include novel controls, such as antitermination and competition between repressors and antirepressors to determine which operons will be expressed.
Lytic and lysogenic infections: Extensive studies have been done on the control mechanisms utilized by bacteriophage lambda as it switches back and forth between lytic and lysogenic phases. These studies are analyzed in far more detail in MCDB 3500 than in our textbook. In order to avoid excessive overlap and because of time limitations, we will not attempt to cover these topics in this course this year. The notes that follow are from past years when we have attempted a quick survey of a few of the more interesting topics. Pages 233-237 of our textbook present a brief summary of this material.
Lytic and lysogenic infections: Bacteriophage lambda was introduced in Chapter 7 (pages 205-206) as an agent for specialized transduction of bacterial cells. We will encounter it again in Chapter 9 (pages 265-268) as a gene cloning vector. As described in Chapter 7, bacteriophage lambda can either undergo an lytic cycle, with sequential stages of gene expression leading to production of new bacteriophage particles and lysis of the host cell, or else it can enter into a lysogenic state, in which the viral genome is integrated into the E. coli host chromosome and carried through many genrations without damage to the host cell. Release from lysogeny generally occurs only when the host cell is under stress, as discussed below.
Defining left and right in lambda phage: Two different ways of defining left and right in lambda phage can be very confusing to the beginner. The lambda genome is a circle, which becomes linearized when packed into a phage head. When one talks of gene expression from the circular viral genome, as is done in this lecture, the point of reference is a central regulatory region, with left and right operons transcribed in opposite directions around the circle from it. The genes needed for lysogeny are in the left operon and the genes for the lytic cycle are in the right operon, including genes for head and tail proteins in the so-called late operon, which is actually an extension of the right operon. However, when the viral genome becomes linearized for packaging in the head of the viral particle, the break occurs in the right operon close to the start of the "late operon. Thus, relative to the ends of the linearized molecule, genes for head and tail proteins are at the left end, and genes for DNA replication and lysis, as well as regulatory genes, are at the right end, with genes needed only for lysogeny in the middle portion (which is replaced with a cloned insert in lambda phage vectors). Upon infection into E. coli, the genome again becomes circular, reuniting the genes for the head and tail proteins with the rest of the right operon.
Antitermination: The bacteriophage lambda genome can be divided into four operons, designated left, right, late (an extension of right), and repressor. During the initial stages of infection, the early genes in the left and right operons are transcribed with host polymerase for only a short distance before a rho-dependent termination occurs in each operon. One of the early products from the left operon is an antiterminator protein coded by the N gene. As it accumulates, it blocks the terminations and allows transcription to spread further into the left and right operons. There is also a second antitermination system involved in the lytic cycle that is not described in our textbook. Transcription of the right operon terminates again, somewhat downstream from a gene designated Q. In cells that enter a lytic cycle, the Q gene product accumulates and functions as another antiterminator, allowing the late operon (actually an extension of the right operon) to transcribe genes needed for completion of the lytic cycle, including those for formation of the phage head and tail. Thus, a series of temporal delays are achieved by forcing further transcription to wait until protein products of earlier transcription events accumulate to levels that are adequate to achieve antitermination. Also, as described below, the second antitermination event fails to occur in lysogeny because transcription of the right operon is blocked.
Choosing lysogeny vs. lysis As indicated above, bacteriophage lambda can either produce a lytic infection or become incorporated into the host genome in a lysogenic state. Ultimately, this reflects the outcome of a race between expression of the product of the cI gene, which promotes lysogeny, and the product of the cro gene, which favors a lytic cycle. In a typical infection of healthy and vigorous E. coli cells, a statistical balance between the two processes occurs, such that most of the cells are lysed, producing a plaque, but a few become lysogenic, giving the plaque a somewhat cloudy appearance. Loss of cI gene function results in clear plaques with no lysogeny (the c stands for "clear"; there are also cII and cIII genes that produce clear plaques when mutated).
Lambda repressor: To achieve lysogeny, bacteriophage lambda must produce a repressor of the lytic functions, which is coded for by the cI gene. The lambda repressor protein, which is the name widely given to the cI gene product, blocks expression of both the left and right operons, and also stimulates transcription of its own message from a repressor maintenance promoter. Thus, once lysogeny has been established, it tends to be self sustaining. In addition, the presence of the repressor protein makes the cell "immune" to lytic infection by other lambda phage. In cases where repression is successful, there is enough transcription of the left operon before it is shut down to generate the integrase enzyme needed for integration of the lambda genome into the host genome. However, transcription of the right operon is shut down before there is enough accumulation of the Q gene product to achieve the second antitermination event that would otherwise activate production of many of the gene products needed for a lytic cycle.
Antirepressor activity: A second gene located somewhat to the right from the cI gene codes for an antirepressor protein, designated cro (for control of repression and other things). The cro gene product inhibits transcription of the repressor operon, including the cI gene, and also has a positive effect on transcription from the left and right operons. In cases where lysogeny is not achieved quickly, the cro gene product accumulates sufficiently to turn off the cI gene (coding for the lambda-repressor) and firmly establish a lytic cycle. The textbook describes the ability of the cI protein to stimulate transcription of its own gene by binding to operator sites located between the cI and cro genes (figure 8.19b). If too much of the cI gene product accumulates, binding to a third site turns off cI transcription. The cro protein also has the ability to bind to the same sites with different relative affinities. When cro is preferentially bound, transcription of cI is repressed, while transcription of the right operon, including more cro, is favored (figure 8.19c). The textbook describes this competition in greater detail than summmarized in these notes. You will not be required to learn all of the details until you get to MCDB 3500.
Alternative pathways of gene expression: As described above, during early stages of lambda phage infection, there is a molecular race between expression of the cI gene product, which leads to establishment of lysogeny, and expression of the cro gene product, which leads to establishment of a lytic infection. In healthy cultures of bacteria, most of the cells are lysed and only a few become lysogenic. Several additional lambda genes, including cII and cIII are involved in this interaction, which is too complex to explore in detail here. One of the key observations, mentioned briefly in the textbook, is that the cII gene product is needed for initial activation of transcription of the cI gene, which then becomes self-sustaining, as described above. In addition, the cII gene product is unstable alone, with the cIII gene product helping to stabilize it. Loss of function of either of these proteins results in lysis. The designation "c" refers to "clear" plaques, in which there are no residual lysogenic cells.
Escape from lysogeny: The lysogenic state allows the viral genome to be maintained indefinitely as a part of the bacterial genome. Continual production of the lambda repressor provides the cell with protection against further infection by lambda phage, as well as some other bacterial viruses. Events that damage the cell often cause a release from the lysogenic state. For example, exposure to ultraviolet light induces the SOS response (pages 145-147), which includes the formation of a protease (activated RecA) that cleaves the lambda-repressor. When this happens, self-stimulation of cI transcription is lost, as is the block to left and right operon transcription, including that of the cro gene. This results in a switch to the lytic pattern of gene expression, as well as excision of the lambda genome from the bacterial chromosome and a new cycle of viral proliferation and cellular lysis.