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Prescott−Harley−Klein: Microbiology, Fifth Edition VI. The Viruses 17. The Viruses: Bacteriophages © The McGraw−Hill Companies, 2002 the second -subunit receives an ADP-ribosyl group and this turns off some of the early T4 genes. The product of one early gene, motA, stimulates transcription of somewhat later genes, one of which produces the sigma factor gp55. This sigma factor helps RNA polymerase bind to late promoters and transcribe late genes, which become active around 10 to 12 minutes after infection. It is clear from the sophisticated control of RNA polymerase and the precise order in which events occur in the reproductive cycle that the expression of T4 genes is tightly regulated. Even the organization of the genome appears suited for efficient control of the life cycle. As can be seen in figure 17.6, genes with related functions—such as the genes for phage head or tail fiber construction—are usually clustered together. Early and late genes also are clustered separately on the genome; they are even transcribed in different directions—early genes in the counterclockwise direction and late genes, clockwise. Since transcription always proceeds in the 5′ to 3′ direction, the early and late genes are located on different DNA strands (see sections 11.5 and 12.1). Considerable preparation is required for synthesis of T4 DNA because it contains hydroxymethylcytosine (HMC) instead of cytosine (figure 17.7). HMC must be synthesized by two phageencoded enzymes before DNA replication can begin. After T4 DNA has been synthesized, it is glucosylated by the addition of glucose to the HMC residues. Glucosylated HMC residues protect T4 DNA from attack by E. coli endonucleases called restriction enzymes, which would otherwise cleave the viral DNA at specific points and destroy it. This bacterial defense mechanism is called restriction. Other groups also can be used to modify phage DNA and protect it against restriction enzymes. For example, methyl groups are added to the amino groups of adenine and cytosine in lambda phage DNA for the same reason. The replication of T4 DNA is an extremely complex process requiring at least seven phage proteins. Its mechanism resembles that described in chapter 11. Restriction enzymes and genetic engineering (pp. 320–21) 386 Chapter 17 The Viruses: Bacteriophages Hydroxymethylase Head filling polymerase RNA Head, neck, and modification Endolysin Tail tube Scaffolding protein DNA ligase Endonuclease rll (lysis) mot Head Tail baseplate and collar m Nucleotide etabolism Tail fiber Membrane T4 DNA synthesis, replication, DNA exonuclease DNA polymerase proteins Tail baseplate Figure 17.6 A Map of the T4 Genome. Some of its genes and their functions are shown. Genes with related functions tend to be clustered together. CH2 OH NH2 O N H N Figure 17.7 5-Hydroxymethylcytosine (HMC). In T4 DNA, the HMC often has glucose attached to its hydroxyl
I VL The Viruse we 172 Reproduction of Double-Standed DNA The Lytic Cyde 31 A B C D E F G Y ZA B G D A B C D E P G Y ZA B C D Exonuclease activity A B C D E F G YZAB C D n each u AB C DEFG HI// T4 DNA shows what is called ter Y Z of several enzymes (figure 17.8).These very long DNA strands C DEFGHI / Y ZAB GH'T H YZABCDEF cleaved in such a way that the genome is slightly longer than the //YZABCD r /YZARCDEEGH is the same bu DNA were DNA circles would have iden tical gene sequences. The Assembly of Phage Particles m) begin ut at an gointin
Prescott−Harley−Klein: Microbiology, Fifth Edition VI. The Viruses 17. The Viruses: Bacteriophages © The McGraw−Hill Companies, 2002 T4 DNA shows what is called terminal redundancy; that is, a base sequence is repeated at both ends of the molecule (figure 17.8). When many DNA copies have been made, about 6 to 10 copies are joined by their terminally redundant ends with the aid of several enzymes (figure 17.8). These very long DNA strands composed of several units linked together with the same orientation are called concatemers. During assembly, concatemers are cleaved in such a way that the genome is slightly longer than the T4 gene set. The genetic map is therefore drawn circular (figure 17.6) because T4 DNA is circularly permuted (figure 17.9). The sequence of genes in each T4 virus of a population is the same but starts with a different gene at the 5′ end. If all the linear pieces of DNA were coiled into circles, the DNA circles would have identical gene sequences. The Assembly of Phage Particles The assembly of the T4 phage is an exceptionally complex selfassembly process. Late mRNA, or that produced after DNA replication, directs the synthesis of three kinds of proteins: (1) phage structural proteins, (2) proteins that help with phage assembly without becoming part of the virion structure, and (3) proteins involved in cell lysis and phage release. Late mRNA transcription begins 17.2 Reproduction of Double-Stranded DNA Phages:The Lytic Cycle 387 Y′ Z′ A′ B′ C′ D′ E′ F′ G′ Y′ Z′ A′ B′ C′ D′ E′ F′ G′ Y′ Z′ A′ B′ C′ D′ E′ F′ G′ A B C D A′ B′ C′ D′ A B C D E F G Y Z A′ B′ C′ D′ E′ F′ G′ Y′ Z′ Exonuclease activity E F G Y Z E′ F′ G′ Y′ Z′ A B C D A′ B′ C′ D′ Y Z A B C D E F G Y Z A B C D E F G Y Z A B C D E F G Figure 17.8 An Example of Terminal Redundancy. The gene sequences in color are terminally redundant; they are repeated at each end of the DNA molecule. This makes it possible to join units together by their redundant ends forming a concatemer. For example, if the 3′ ends of each unit were partially digested by an exonuclease, the complementary 5′ ends would be exposed and could base pair to generate a long chain of repeated units. The breaks between terminal sequences indicate that the DNA molecules are longer than shown here. A′ B′ C′ D′ E F G H I A′ B′ C′ D′ A B C D E F G H I Y Z E′ F′ G′ H′ I′ Y′ Z′ C D E F G H I Y Z A B G′ H′ I′ Y′ Z′ A′ B′ C′ D′ Y Z I′ Y′ Z′ A B C D E′ F′ E′ F′ G′ H′ Y Z A B C D E F G H I Y′ Z′ A′ B′ C′ D′ E′ F′ G′ H′ I′ Figure 17.9 Circularly Permuted Genomes Cut from a Concatemer. The concatemer formed in figure 17.8 can be cut at any point into pieces of equal length that contain a complete complement of genes, even though different genes are found at their ends. If each piece has single-stranded cohesive ends as in figure 17.8, it will coil into a circle with the same gene order as the circles produced by other pieces