BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 30. VIRUSES

30.5. Accessory Proteins and Proteases Participate in the Ordered Assembly of Phage T4

The assembly of phage T4 is a significantly more complex process than that of TMV, as the T4 capsid possesses a much more intricate structural Organization and contains approximately 40 different Proteins. An additional thirteen auxiliary proteins (compared to TMV) take part in phage T4 assembly without being Structural components of the capsid. The assembly mechanism of this virus has been elucidated through a combination of genetic, biochemical, and electron microscopic Methods. Work by William Wood and Robert Edgar on assembly-defective mutants of phage T4 established the following key findings.

1. There are three Main Pathways of reactions that lead to The formation of the virus. Through these pathways, the HEAD, the tail, and the tail fibers are formed independently (Fig. 30.11). Blocking the formation of any one of these components does not affect the Synthesis of the other two.

Class="center">Fig. 30.11. Morphogenesis of phage T4. Numbers near the arrows designate genes whose products are required for the corresponding stages of assembly

2. Each of these reaction sequences proceeds in a strictly determined order. All capsid proteins are synthesized simultaneously in the latter half of the infection cycle. Thus, the structural Properties of the intermediate products themselves enforce the strict sequence of head, tail, and tail fiber assembly. None of these association processes can proceed at a noticeable rate until the preceding one is complete. Part of the binding energy at each stage is likely utilized to lower the activation energy of the subsequent association process, thereby increasing its rate.

3. The head and the tail must be fully assembled before they join one another. Subsequently, the preformed tail fibers attach to the baseplate. Here too, the strict sequence of events ensures that only complete, mature Viral Particles are released.

The formation of phage T4 virions does not rely solely on self-assembly. Auxiliary (morphopoietic) proteins and proteases play a crucial role at certain Stages of the process. For example, the Formation of the central "tube" of the tail baseplate requires three proteins that are not part of the assembled tail. These accessory proteins serve as temporary templates for the association of the tube components. Proteases play a vital role in head assembly. The major head protein with a mass of 45 kDa, designated gp23* (where gp stands for Gene product), is formed from a 55 kDa precursor called gp23. Cleavage occurs when the head is partially assembled, indicating that it triggers the mechanism for DNA translocation into the head. Three additional head proteins are known to be cleaved during assembly. Thus, phage T4 is formed through a combination of self-assembly and assembly mediated by accessory (morphopoietic) Proteins and Enzymes.

30.6. A Concatemeric Intermediate Participates in Phage T4 Replication

The Replication of linear DNA molecules, particularly phage T4 DNA, presents a specific problem. The 5'-ends of the newly synthesized daughter DNA are incompletely filled in because the RNA primer was removed but not replaced by DNA (Fig. 30.12). Recall that DNA polymerase is unable to synthesize DNA chains de novo in the 3' → 5' direction (Section 24.19). This problem does not arise during the replication of circular DNA molecules, since the 3'-end of the new strand serves as a primer to complete the synthesis of the daughter strand. How do phage T4 and other Viruses with linear DNA genomes solve this problem? An important clue came from the discovery that these linear molecules exhibit terminal redundancy, meaning The base sequence of the left end of the DNA is exactly repeated at the right end:

Fig. 30.12. The 5'-ends of newly synthesized linear DNA molecules are incompletely filled in. Parental DNA strands are shown in red, and daughter strands in blue

Furthermore, the replication of these DNA molecules yields long concatemers. These discoveries led to a proposed mechanism for filling in the 5'-ends of the daughter strands. Because the single-stranded ends of the newly formed double-stranded molecules are mutually complementary, they rapidly reassociate (Fig. 30.13). This complementarity is a consequence of terminal sequence redundancy. In the concatemeric chain, which consists of repeating units of double-helical phage DNA molecules, the 3'-end of one molecule serves as a primer to fill in the 5'-end of another.

Fig. 30.13. Concatemeric intermediate in the replication of linear double-stranded DNA molecules. Double-stranded DNA molecules with unfilled ends associate with one another through the pairing of complementary single-stranded ends (AB with ab). The single-stranded gaps are then filled in

30.7. Phage T4 DNA is Inserted into a Preformed Head

How is a DNA molecule corresponding to a single phage genome generated, and how is it packaged into the head? This is an immensely challenging problem: the DNA has a contour length of 56 µm and yet must fit inside a head whose major axis is only 0.1 µm long. Moreover, the volume of the DNA (1.8 • 10^-4 µm³) is not much smaller than the volume of the head (2.5 • 10^-4 µm³). A priori, There are two possibilities: DNA could enter a preformed head, or the head could assemble around a "core" of condensed DNA. The isolation of empty phage heads capable of packaging DNA provides direct proof that phage T4 DNA is inserted into preformed heads (Fig. 30.14). As the DNA enters the head and condenses into a Structure resembling a ball of yarn, several head proteins undergo cleavage.

Fig. 30.14. Scheme of DNA packaging during phage T4 head assembly

Simultaneously, the head undergoes Swelling. Finally, once a DNA fragment corresponding to the length of a single genome has entered the head, the concatemeric DNA is cleaved. The nuclease acts not by recognizing a specific sequence, but by cleaving the DNA precisely when the head is full. This explains why the ends of T4 DNA molecules exhibit terminal redundancy; the DNA is packaged in such a way that it can be very rapidly injected into the bacterium during the next infection cycle. Just how this remarkable mobility is achieved remains a mystery.