Question

3. The lysogenic bacteriophage lamda uses a variety of proteins and promoter/operator regions to determine course of action (lytic or lysogenic). Choose any five of the following genes, proteins or regulatory sequences and write a brief description of their function in the 'decision' process of this virus:

a) PRM

b) c2

c) N

d) Q

e) Int

f) Xis

Answer 1

a) PWM- Bacteriophage λ is a temperate bacteriophage, meaning that it can reproduce and develop either in a lytic or lysogenic state. Although very stable, the lysogenicor prophage state can be reverted by inducing agents that damage the host DNA, returning the virus λ to its lytic state.

b) C2 - Enterobacteria phage λ (lambda phage, coliphage λ, officially Escherichia virus Lambda) is a bacterial virus, orbacteriophage, that infects the bacterial species Escherichia coli. However, under certain conditions, the phage DNA may integrate itself into the host cell chromosome in the lysogenic pathway.

c) N - The functional half-life of the phage N gene product, measured by complementation, is increased about 5-fold in Ion mutant strains, from 2 min to 10 min. The chemical half-life of N protein, determined by its disappearance on polyacrylamide gels following pulse-chase labeling, increases about three-fold in Ion cells. In contrast to its effect on the N protein, the Ion mutation produces a 50% decrease in the chemical half-life of cll protein. The decay rate of many other phage proteins, including the unstable gene O product, remains unaffected by a host Ion defect. A Ionmutation alters lambda physiology in two ways. First, upon infection, the phage enters the lytic pathway predominantly. This may result from the deficiency of cll protein caused by its decreased stability, since cll product is required for establishment of lysogeny. Second, brief thermal induction of a Ion lysogen leads irreversibly to lysis; repression cannot be restablished and the treated cells are committed to forming infective centers. Although N product is normally required for rapid commitment, Ion lysogens become committed more rapidly than Ion lysogens, even in the absence of Nfunction. These results identify for the first time native proteins whose stability is affected by the Lon proteolytic pathway. They also indicate that the Lon system may be important in regulating gene expression in E. coli.

e) Int -The integration excision reaction as a crucial aspect of the choice of pathway during lysogenic or lytic viral development. This control involves differential expression of the tightly linked, partially overlapping int and xis genes from two promoter sites: pI, positively regulated by cII/cIII proteins, and pL, positively regulated by N protein. After lambda infection, Int is synthesized from the pI transcript under cII regulation; however, very little Int is produced from the pL RNA because of the existence of a cis-acting regulatory element, sib, on the opposite side of the int gene from the pL promoter. Presumably sib serves to prevent unwanted synthesis of Int protein during the lytic response; the Int protein necessary for excisive recombination from a prophage can be supplied by pL transcription because sib is separated from int by prophage insertion. We have studied the effect of sib on nearby lambda genes by means of gel electrophoresis of labeled proteins from infected cells. Deletion of the sib region greatly enhances production of Int protein without substantial effect on Xis production; thus, sib regulation normally is highly specific for Int. When the sib region is moved past int and xis by deletion, regulation of the adjacent gene for the protein Ea22 occurs, suggesting that sib regulation can work on other genes. Although synthesis of wild-type Int is severely inhibited by sib, shorter Int protein fragments generated by nonsense mutations escape sib regulation, indicating that the regulation is translational and occurs near the completion stage of protein synthesis. Regulation by sib thus exhibits novel regulatory features: distal location, recombinational control, and regulation of the completion of protein synthesis. Because Int and Ea22 control is lost in a RNase III- host, we suggest that sib regulation might involve RNase III cleavage of a RNA duplex region that includes sib and the regulatory target (normally the int gene). We note such a potential site within int.

f) Xis - Bacteriophage lambda (l) The phage genome integrated into the host bacterial genome is a prophage. Lysogens are immune to further infection by similar phagebecause the phage functions are repressed in trans.