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Terms in this set (15)

During phage T7 infection in E. coli, early and late genes are
expressed essentially in the order in which they are arranged on the viral genome. The early genes comprise the left-most 20% of the phage's genome and the late genes comprise the right-most 80% of the genome.

It turns out the early genes are transcribed by the bacterial host cell RNA polymerase. Phage T7 late genes are transcribed by a phage-encoded RNA polymerase called GP1 (or T7 RNA Polymerase) that is totally unrelated to the bacterial host RNA polymerase. The phage polymerase, "T7 polymerase", is the product of an early gene, called gene 1. The T7 polymerase is composed of a single polypeptide chain and is specific for the phage late genes because it only recognizes the late promoters. It takes ~8 minutes to transcribe the gene 1 gene and translate the gene 1 mRNA to make functional T7 polymerase proteins.
**Phage T7 just tries to make as many phages as possible (170nt/sec), doesn't care about proofreading. Goes much faster than E. coli RNAP (20-50 nt/sec).

Transcription of early genes (and host genes) ceases
during late gene expression. The product of the late gene 2 GP2 binds to E. coli host RNA polymerase holoenzyme and totally inhibits its initiation/activity. This inactivation results in shutting off all RNA synthesis from bacterial host and early phage promoters. Twelve minutes after phage infection, the only transcription occurring is from the late phage promoters, signifying a complete take-over of the cell's metabolic processes by the bacteriophage.

**Transcription termination of early genes is both factor independent (encoded termination site) and factor dependent (GP2 from late genes inhibit host RNA polymerase)
How does the virus decide whether to replicate itself and kill its host cell or to shut off the lytic cycle in order to form a stable relationship with the cell? E. coli bacteriophage λ is the classical model for understanding the decision process (and also the ancestor of the phage that causes hemolytic-uremic syndrome).

A. Lytic growth of λ
- λ is a double-stranded DNA virus containing about 50 genes. Following infection of the cell, the ends of the linear virus genome come together, forming a closed circle. Transcription of the viral genes occurs in three phases, all of which require E. coli host RNA polymerase. Key sites are the left and right promoters, and late promoters, depicted as PL, PR, and Plate, respectively.

EARLY gene expression - initiates at PL and PR and ends at terminators found downstream of the promoters. Early gene expression leads to the production of the N protein.

MIDDLE gene expression - initiates from PL and PR but requires the N protein, which acts as a transcriptional "antiterminator," allowing RNA polymerase to bypass the early terminators because N protein binds to the terminator site and blocks termination, allowing for the production of longer transcripts. In the presence of N protein, leftward transcription proceeds through the int gene; rightward transcription proceeds through the Q gene. In addition to the Q protein, middle genes encode phage DNA replication proteins (O, P), integration and excision proteins (Int, Xis), and the regulatory protein CII.

LATE gene expression - initiates from Plate promoter just upstream of late genes and requires the Q protein (encoded in middle RNA) to act as an antiterminator of transcription from Plate. Late genes encode phage structural components and the host lysis enzymes.
***The only gene not transcribed in the lytic cycle is the cI gene, which is the repressor of lytic growth. If cI protein is activated, it represses lytic growth, need two promoters that were previously not active to become functional, PRM and PRE.
When phage λ infects a cell that is growing slowly (for instance, a cell growing on a poor carbon source), the phage opts to repress itself rather than try to make any progeny phage and lyse the host cell. Under poor growth conditions, the phage has evolved mechanisms to shut itself off before committing to replication and destruction of the host cell-the result of initiating late gene expression. To choose the lysogenic response, the phage needs to do two things quickly: synthesize a high concentration of CI repressor and synthesize the Int protein, a DNA recombination protein, in order to integrate the phage genomic DNA into the bacterial host chromosome. The response to the nutritional status of the host cell is mediated through the phage regulatory protein, CII, and a host protease that uses CII as a substrate.

i) Initial synthesis of the λ repressor, CI, depends on the presence of the CII protein, a transcriptional activator that binds to PRE, the "promoter for repressor establishment."
During an infection that will lead to lysogeny, the early and middle stages of phage gene expression occur in exactly the same manner as for a lytic infection. If environmental conditions promote lysogeny, the CII protein builds up and binds at PRE, acting as positive regulator, leading to high levels of CI repressor expression. The CI repressor binds immediately to PL and PR, shutting off all early and middle gene expression of the phage, thereby preventing lytic infection.

ii) Maintenance of repression is due to CI binding at PRM. Once repression is established (PR and PL are shut off) there is no more synthesis of CII.Thus, the concentration of CII protein will diminish as the host cells grow and divide, and there will no longer enough CII to stimulate expression at PRE. In order to maintain repression of the lytic cycle, phage λ has a second mechanism for synthesizing the repressor, another promoter, PRM. This promoter requires the λ CI repressor to act as a positive regulator for its own synthesis. PRM can only be used when there is already repressor protein in the cell. The location of PRM is interesting as it is within the PR site, but drives transcription
in the opposite direction of PR.

iii) Whether a phage initiates lysogeny depends on the nutritional status of the cell. The CII protein is exquisitely sensitive to a certain host protease whose levels fluctuate in
response to the host's nutritional status. When cells are growing in rich medium, there is high expression of a protease and the CII protein is quickly degraded. In the absence of CII, there is no activation of the PRE promoter and the phage can enter into the lytic cycle. Conversely,
when the cells are growing in a poor medium, the is very little protease sparing CII, allowing it to activate transcription at PRE and express the λ repressor, CI. As a result, lysogeny ensues and the phage genome integrates into the bacterial chromosome.

POOR MEDIUM -> LOW PROTEASE AND Q VIA ACTIVATED CII-> Phage undergoes LYSOGENY
RICH MEDIUM -> HIGH PROTEASE AND Q VIA INACTIVATED CII-> Phage undergoes LYTIC GROWTH
A bacteriophage is an obligate parasite with no metabolism of its own, and thus its goal to reproduce itself is at the expense of its host. Phage are the most abundant biological entity on earth outnumbering bacteria 10-100 fold. Therefore it is of selective advantage for a bacterium to become resistant to phage infection. There are many strategies that the bacterium can use to defend itself against viral attack, in essence to become resistant to the bacteriophage. Three examples are listed below.

1) The bacterial host can evolve by altering its surface proteins that are used by a virus to adsorb. The altered proteins would no longer be recognized as receptors by the virus.

2) Degradative endonucleases, called restriction enzymes can cleave or "restrict" phage DNA genomes. The host genome is protected because it is modified. Host restrictionmodification systems are found throughout the bacterial world. For example: E. coli DNA is methylated and it produces an enzyme that cuts any foreign (unmethylated) DNA that enters the cytoplasm. Purified restriction enzymes are used for genetic engineering.

3) A bacterial mechanism of adaptive immunity against invading nucleic acids (e.g. from phage, conjugation, etc.), called CRISPR has been recently discovered. CRISPR systems have been found in over half the bacterial species. Bacteria with CRISPR immunity will specifically cleave foreign DNA at specific sites with surgical precision using a CRISPR endonuclease and an RNA guide. About 50% of bacteria have CRISPR system; CRISPR locus contains repeat sequences between all spacer DNA sequences that match with a phage DNA sequence. Each spacer matches with a different bacteriophage. Upstream of CRISPR locus is cas genes (palindromic), cas genes make endonuclease that can cut DNA. Cas endonuclease will lead to stem structures that have specific bacterial spacer DNA. If piece of DNA matches, double sided cleavage takes place and prevents infection. In lab can design guide RNAs to give cut and break target genes, can then deliver good genes through cas system to replace broken gene with good gene. The CRISPR system is currently being actively studied for genetic engineering and has huge implications