The Structure and Genetic Map of Lambda phage

Transcription

1 NPTEL Biotechnology - Systems Biology The Structure and Genetic Map of Lambda phage Dr. M. Vijayalakshmi School of Chemical and Biotechnology SASTRA University Joint Initiative of IITs and IISc Funded by MHRD Page 1 of 8

2 NPTEL Biotechnology - Systems Biology Table of Contents 1 FLUCTUATIONS IN GENE EXPRESSION... ERROR! BOOKMARK NOT DEFINED. 1.1IS NOISE AN ADVANTAGE TO BIOLOGICAL SYSTEMS?... ERROR! BOOKMARK NOT DEFINED. 1.2 MEASUREMENT OF EXPRESSION NOISE... ERROR! BOOKMARK NOT DEFINED. 2 REFERENCE... ERROR! BOOKMARK NOT DEFINED. 2.1 LITERATURE REFERENCES... ERROR! BOOKMARK NOT DEFINED. 1.2 VIDEO LINK... ERROR! BOOKMARK NOT DEFINED. Joint Initiative of IITs and IISc Funded by MHRD Page 2 of 8

3 1 Introduction Viruses are obligate intracellular parasites. Bacteriophages are viruses infecting bacteria. They multiply inside the host system through partial or complete utilization of the host biosynthetic machinery. Bacteriophages may be RNA phages such as Q-beta Filamentous with single stranded DNA such as M13 T- even phages including T2,T4,T6 infecting E.coli Temperate phages like lambda and mu Spherical phages with single stranded DNA like PhiX174 The genetic material in bacteriophage may either be RNA or DNA but not both. The nucleic acids of phages contain unusual or modified bases which enable to circumvent the degradation of the host nuclease. Further, the number of genes in bacteriophage varies with the complexity of the genome. Complex phages have more than 100 genes while simple phages have only 3-5 genes. 1.1 Structure of the Lambda Phage Lambda phage is a temperate phage. The genes of the lambda phage make a single DNA molecule-the chromosome wrapped within a protein coat, composed of different proteins all of which are encoded by the lambda chromosome. The coat is structurally composed of an icosahedral head with a diameter of 64nm and a tail, 150 nm in length as shown in Fig 1. The head is composed of double stranded linear DNA surrounded by a capsid made up of protein capsomers. At the 5 end of each strand are 12 nucleotide long sequences complementary to each other. Thus on circularization, the bacteriophage DNA has 48,514 base pairs. The 5 ends are called cos sites and the opposite of cos site is att site meant for attachment. The lambda phage attaches to the cell surface of E.coli through its tail, making a hole in the cell wall. It thus pushes its chromosome into the bacterium E.coli, leaving behind the protein coat. Joint Initiative of IITs and IISc Funded by MHRD Page 3 of 8

4 Fig 1 (a) Structure of a Lambda phage, (b) measurement of different parts of a Lambda phage First stage of infection involves a process called adsorption. Adsorption involves landing and attachment. Tail fibres play a critical role in this stage. Tail less phages use analogous structures for adsorption. Specific receptors on the bacterial cell like proteins, lipopolysaccharides, pili apart from lipoproteins are exploited by phages for attachment. This is reversible condition. Base plate components mediate permanent binding. Second stage in infection process is penetration. The sheath of phages contracts resulting in insertion of hollow tail fiber through bacterial envelope. Some phages utilize their enzymes to digest components of bacterial envelope. Nucleic acid is inserted inside bacterial cell via hollow tail. Remaining part of the phage outside bacteria is called ghost. Thus in nutshell penetration involves contraction of sheath till DNA insertion. Some phages upon infecting bacteria lyse the bacterial cell after forming their progeny. Such phages are called virulent. Some phages integrate their genome into bacterial genome and can remain inside host without harming them but under drastic conditions can become virulent and can causes host cell lysis. Such phages which normally follow lysogeny but under drastic conditions become lytic are called temperate phages. Joint Initiative of IITs and IISc Funded by MHRD Page 4 of 8

5 1.2 Receptor targeting λ- phage The λ - phage uses the maltose pore LamB for delivering its genetic material into the host cell. The phage binds to the cells of the target E.coli and the J-protein in the tip of its tail interacts with the LamB, gene product of E.coli (LamB is a porin molecule and is a part of the maltose operon). Most of the E.coli K-12 mutations resistant to λ phage are located in two genetic regions mala and malb. LamB is composed of three identical subunits, each of which is formed by an 18-stranded antiparallel β-barrel, which forms a wide channel with a diameter of about 2.5 nm. The phage consists of a hollow tube composed of 32 stacked discs, each of which has a 3nm central hole to eject its genetic material into the host. Lambda phage uses this channel for ejecting its genetic material. After injecting the DNA into bacteria, the double stranded linear DNA circularizes due to the presence of cos sites and site specific nucleases cut DNA at the att site of the phage DNA. Fig 2. Genetic mapping of lambda phage Joint Initiative of IITs and IISc Funded by MHRD Page 5 of 8

6 Table 1 The genes involved in the switching mechanism of the lambda phage Name of gene/promotor/operator Function Att ci Provides site of attachment for phage to host chromosome Repressor protein cii Coding for promotor establishment activator protein ciii OR PR OL PL Cro Codes for stabilizing protein Operator right Promotor right Operator left Promotor left Gene for second repressor N Positive regulator counteracting rho dependent termination J through U Z through A Int Xis O and P Q Genes encoding tail proteins Genes encoding head proteins Gene encoding integrase Encodes excisionase Encode proteins involved in replication of Lambda DNA Encodes anti terminator protein Joint Initiative of IITs and IISc Funded by MHRD Page 6 of 8

7 Bacteriophage lambda is episomic and consequently its genome exists in at least two states within which genetic recombination is possible. This allows the construction of two genetic maps termed vegetative and prophage after these states. In the vegetative state, the replication of the lambda genome is independent of the replication of the host genome. Such replica are finally packaged into the head of the mature phage as single duplex DNA molecules, 15 to 17 microns in length. These molecules contain ~ 47,000 base pairs, to accommodate 40 to 45 structural genes. In the prophage state the viral genome integrates into the host genome replicating in synchrony with the host genome. Lambda genes are organized into operons. The Left operon genes are meant for recombination and integration resulting in lysogeny, while right operon and the late operon genes are meant for lysis. The genetic map of the lambda phage is shown in Fig2. The lambda phage infects the bacterium directing it to two different fates. In some of the cells, as the infection happens the different set of phage genes are turned ON and OFF in a precisely regulated manner. The lambda chromosome is replicated, newer head and tail proteins are synthesized, forming new phage particles within the bacterium. As the phage chromosome begins to replicate, the phage gene ci, is expressed. The product of ci is the bacteriophage lambda repressor which keeps the other phage genes in the OFF state. When exposed to ultraviolet light, the inert phage genes (lytic genes) are switched ON and the repressor gene is switched OFF. Nearly 45 minutes after the infection the bacterial cell lyses releasing around 100 new progeny phage. In the other population of cells, the injected phage chromosome turns OFF all the phage genes except one. The single phage chromosome called the prophage now becomes a part of the host chromosome. The bacterium carrying the dormant phage chromosome is called the lysogen. As the lysogen grows, the prophage is passively replicated with the host genome and distributed to the progeny bacteria. Thus, we understand that the phage genes upon exposure of a lysogen to a signal such as UV, switch from their stable lysogenic state to a lytic growth state. The switch from the lysogeny to the lytic growth is termed induction. We shall discuss the classic switch of a lambda phage in the next class. Joint Initiative of IITs and IISc Funded by MHRD Page 7 of 8

8 2 References 2.1 Text Book 1. A Genetic switch: Phage lambda revisited, 3/e, CSHL Press, New York, (2004). 2.2 Literature References 1. DAVID S. HOGNESS et al., The Structure and Function of the DNA from Bacteriophage Lambda. The Journal of General Physiology,(1966), 49, Oppenheim A B, Oren Kobiler, Joel Stavans, Donald L. Court, and Sankar Adhya. Switches in bacteriophage Lambda development, Annu. Rev. Genet., (2005), 39, Fogg P C M, Allison H E eta al, Bacteriophage lambda: A paradigm revisited, J.Virol, (2010), 84, Video Link Joint Initiative of IITs and IISc Funded by MHRD Page 8 of 8