Please note that classroom presentation of this material will
be limited to the time available at the end of lecture 35 on the
trp operon. Bacteriophage lambda is a fascinating system that
is well covered in MCDB 3500. We will not have time this year to
discuss it in detail. I have left the notes on it in place for those
who may be interested. However, bacteriophage lambda
will not be covered on the final beyond those items that
we have time to discuss in lecture plus items mentioned earlier in
the semester.
Added 12/2/98: Specifically, there will not be any
questions about
the molecular mechanisms of lysogeny versus a lytic cycle, as indicated
below in this set of notes and in the review questions. However,
antitermination may be included on the examination.
Textbook assignment: Chapter 19, pages 534-543 (includes end of chapter material for entire chapter). You should also read the section on "Differential Transcription in Development: Prokaryotes" on pages 568-569. Material on Lysogeny vs. Lysis in bacteriophage lambda on pages 534-536 will not be included on the examination due to lack of detailed coverage of that topic in lecture.
Major concepts:
Alternative sigma subunits: The sigma subunit (sigma factor) determines the transcriptional specificity of prokaryotic RNA polymerases. Virally-coded sigma factors often alter the specificity of host RNA polymerase during bacteriophage infections. Alternative sigma factors are also used to alter transcriptional specificity during bacterial sporulation and in other phenomena, such as the bacterial heat-shock response, that we do not have time to explore in this course.
Bacteriophage SPO1 infection of Bacillus subtilis: Transcription of the SP01 viral genome can be divided into three time periods, early, middle, and late, based on how much time elapses after infection before the genes in question begin to be transcribed. Early transcription uses host cell sigma factor. A gene coding for an alternative sigma factor, designated gp28, is transcribed during the early period. As it accumulates, gp28 replaces host sigma factor, causing transcription of host genes to stop and transcription of "middle" genes to begin. Middle transcription includes mRNAs for gp33 and gp34. A complex of gp33 + gp34 replaces gp28 as the transcription specificity factor, causing a shift to transcription of late viral genes (see figure 18.12).
Bacterial sporulation: Bacillus subtilis can form highly resistant spores in response to adverse conditions. The sigma factor that is employed during vegetative growth has a molecular weight of 43 kilodaltons. During sporulation, three new sigma factors are made with molecular weights of 29, 30, and 32 kDa. Each of these sigma factors recognizes a set of promoters with different -10 and -35 sequences. As a result of these changes, a very different set of genes is expressed during sporulation. This process is described on pages 568-569 in chapter 20 of our textbook.
Viral-specific RNA polymerase: Some types of viruses employ a newly synthesized virally-coded RNA polymerase for the transcription of viral genes. As an example, after bacteriophage T7 infects E. coli, there is a temporal shift in transcription from early (Class I) genes to later (Class II and III) genes. The Class I genes are transcribed with host RNA polymerase. One of the early gene products is a new RNA polymerase that is highly specific for transcription of Class II and III genes of bacteriophage T7. One of the Class II gene products inactivates the host RNA polymerase, thus completing the switch from host-specific to T7-specific transcription.
Bacteriophage lambda: Bacteriophage lambda was introduced in Chapter 6 as an agent for genetic transduction (pages 165 and 167-168), and again in Chapter 15 as a cloning vector (pages 434-435). 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.
Complexity of lambda phage gene expression: The overall complexity of the gene regulatory mechanisms that occur in bacteriophage lambda is far greater than indicated in the textbook and far beyond what can be discussed in this course. Several lectures in MCDB 3500 will be devoted to the major aspects of the regulatory mechanisms in lambda. Ultimately, whole books have been written on the subject (see Hershey, 1971 and Ptashne, 1986, in the reading list of page 543).
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. 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). However, upon infection into E. coli , the genome becomes circular. When one talks of gene expression from the circular viral genome, the point of reference is a regulatory region, with left and right operons transcribed in opposite directions around the circle from it. This places the genes for lysogeny in the left operon, rather than cental, and the genes for head and tail proteins in the so-called late operon, which is an extension 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. 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.
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 cases where repression is successful, there is enough transcription of the left operon before it is shut down to generate the gene products 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 a pair of operator sites located between the cI and cro genes (figure 18.11). If too much of the cI gene product accumulates, binding to a third site turns off the transcription. The cro protein also has the ability to bind to the same sites, and when it is preferentially bound, transcription of cI is repressed, while transcription of both the left and right operons, including more cro , is favored.
Alternative pathways of gene expression: Thus, 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.
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 411-412), which includes the formation of a protease (activated Rec A) 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.
Antisense RNA: One interesting control mechanism that is encountered occassionally in nature and with increasing frequency in genetic engineering is the use of antisense RNA to form a double stranded hybrid with messenger RNA (or viral genetic RNA) that blocks the message function of that RNA. The textbook contains a boxed article on pages 538 and 539 about possible use of antisense technology in viral disease control. In addition, antisense RNA technology was also used to block expression of the polygalacturonase gene in the Flavr Savr tomato (boxed article on pages 12-13).