Bacteriophage T4

T4 is a bacteriophage that infects Escherichia coli bacteria. The T4 phage is a member of the T-even phages, a group including enterobacteriophagesT2 and T6.

It is one of the largest phages, encoding roughly 200 genes and was the first prokaryotic organism providing evidence of gene splicing through presence of introns in the genome. T4 is capable of undergoing only a lytic lifecycle and not the lysogenic lifecycle.

History of T4.

T4 has been extensively studied and has a rich history in the advancement of genetics. Some of the first essential ideas of genetics came from studies using T4 including: the basis of genetic code, even ribosomes, mRNA, and the codon. It is surprising that science has learned so much using a virus which is extremely complex. Nonetheless, we owe much of our current knowledge to T4.

Genome and Structure

The elongated isocahedral head of T4 phage contains linear double-stranded DNA genome about 169 kbp long and encodes 289 proteins including two accesory proteins, HOC (highly antigenic outer capsid proteins) and SOC (small outer capsid proteins). Binding of SOC stablizes the capsid and resist osmotic shocks.

The T4 genome is terminally redundant and is first replicated as a unit, then several genomic units are recombined end-to-end to form a concatemer. When packaged, the concatemer is cut at unspecific positions of the same length, leading to several genomes that represent circular permutations of the original. The genome is AT-rich and contains modified bases in the form of 5 hydroxy-methyl-cytosine, rather than cytosin, which protect the phage DNA from many host restriction systems and from phage-encoded nucleases that degrade cytosine-containing host DNA during infection. The T4 genome bears eukaryote-like intron sequences.

The T4 tail fibres, pins and base plates are involved in binding to the lipo-polysaccharide receptor of the E.Coli. The T4’s tail is hollow so that it can pass its nucleic acid into the cell it is infecting after attachment. The tail attaches to a host cell with the help of tail fibres.

The life cycle of T4

1. T4 adsorption and Injection

Phage binds to the lipo-polysaccharide and tryptophan receptor on the bacterium. T4 looks for a susceptible bacterium with its tail fibers. The tail fibers recognise the membrane first. The phage once bound to the cell, the base plate goes under conformational change which causes the sheath to contract piercing the cell membrane allowing the viral core to enter the cell, and release the dsDNA viral genome.  The outer sheath contracts driving the internal tail tube in to the cell. The Gene product, gp5 (known as tail lysozome) facilitates digestion of the peptidoglycon layer of the bacterium to reach the inner membrane.

The trans-membrane electrochemical potential is required for transfer of T4 DNA to cytoplasm. The empty capsid remains extracellular.

2. Replication and expression of genes.

Once T4 DNA is in the cytoplasm, it specifies a highly organised and coordinated program of gene expression with the help of 3 promoters; Pe (early genes), Pm (middle gens) and PL (late genes). Some of the first genes which are transcribed, called immediate-early, encode enzymes which break down host DNA. Host DNA is broken down in order to use host nucleotides to produce more viral DNA. It also encode the proteins needed for DNA synthesis and to build the capsid and tail structures.

The host enzyme, RNA polymerase, transcribe and translate the expression of early genes. Early genes rely on the transcription apparatus of the host, being transcribed from normal sigma70 promoters.

Delayed early and middle genes encode the 20 proteins which are involved in viral replication. In fact, T4 encodes nearly all of its own replicative machinery. There are two distinct replicative processes of T4 which are outlined below:

  1. Stage 1 – Replication occurs in a bidirectional manner with multiple origins of replication within the genome. The first several rounds of replication are initiated by RNA primers synthesized by the host RNA polymerase. These RNA primers can travel to their complimentary region in dsDNA and displace the other strand to produce a structure called an R-loop. The attached RNA can now act as a primer for the leading strand of DNA replication. The lagging strand is then synthesized using the replicative helicase, gp41. DNA replication is finished by gp30 which is the T4 encoded ligase as well as the host DNA ligase. Several minutes after infection, host RNA primers cannot be used because the promoting recognition specificity is altered on the host RNA polymerase and recombinant-dependent replication (RDR) is favored. The mechanism of RDR is outlined in stage 2.
  2. Stage 2 – At this point the virus uses its own replicative machinery to transcribe its late genes with a process known as the gp45 sliding clamp model. The primers for leading strand synthesis are recombination intermediates instead of RNAs made by RNA polymerase. 

Early gene products alter the host RNA polymerase in two ways for expression of middle genes. Modifies RNA polymerase will not stop translation and over shoot to reach Pm (middle gene promoter). The enzyme is there by able to read through a transcription terminator by anti-termination mechanism to express genes downstream of early genes and is modofied to recognise middle promoters.

Transcription of the late gene is coupled to replication. Promoters for these genes again differ from sigma70 promoters and require an alternative sigma factor, encoded by T4 regulatory gene, gp55, to activate transcription. Binding of gene, gp55 to host RNA polumerase allows the enzyme to recognise specifically T4 late gene promoters. Other T4 gene products, gp44, gp45 and gp62 are additionally required for late gene transcription.

3. Assembly and Release.

Once late genes are expressed, the viral base plate is first assembled, this then attaches to the tail and tail fiber proteins. These three different protein pathways combine to form a mature T4 phage capsid. DNA is packaged into the mature capsid protein by packaging and cutting the concatemers using a terminase complex found at the end of the concatemerized DNA strand. The terminase complex binds to the capsid head and moves DNA into the empty capsid head. The capsid also encases necessary enzymes for future infections such as virally encoded DNA polymerase.

The viral encoded enzyme holin (gpt) creates the holes in the inner membrane of the bacterial host-cell to allow lysozymes to exit and degrade the peptidoglycan cell wall. Cell lysis subsequently follows releasing a shower of bacteriophage progeny into the extracellular space.

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