Week 4 L1 - Life Cycle of Bacteriophage

Question 1

The five stages of viral replication?

Answer 1

1. Attachment
2. Penetration/Entry
3. Biosynthesis
4. Maturation/Assembly
5. Release

Question 2

What is the difference in replication between T4 and λ, once inside the cell?

Answer 2

• T4 replicates only by the LYTIC CYCLE

* λ is a temperate phage which can replicate by either the LYTIC CYCLE or the LYSOGENIC CYCLE

Question 3

What is transduction? What are the two types?

Answer 3

Transduction is the accidental transfer of bacterial DNA
between species by phages - may be generalised or specialised in nature. Bacteriophages act as vectors and transfer genes from one bacterium (donor) to another (recipient).
—GENERALISED: Random genes may be transferred
between bacterial cells. If the recipient cell is deficient in something and receives a wild type gene from the donor, this is advantageous for the recipient cell.
—SPECIALISED: During the change from the lysogenic to the lytic cycle, every so often by mistake, as the viral genome excises itself from the host genome via homologous recombination in preparation to form particles and get out of there, it takes a chunk of the bacterial DNA with it. Only specific genes are transferred between bacterial cells, on or near the attachment site, because the lambda genome integrates into the bacterial chromosome at a specific site between the gal region and the bio region. The site on the E. coli chromosome is called att-lambda – (Attachment site for lambda). It is homologous with a site called att in the lambda DNA, allowing direct insertion.

Question 4

Promoter

Answer 4

Transcription initiation sequence.

Question 5

Operon

Answer 5

Group of genes transcribed from the same promoter.

Question 6

Repressor

Answer 6

Blocks transcription from the promoter.

Question 7

Operator

Answer 7

The site at which the repressor binds to block transcription.

Question 8

Inducer

Answer 8

Induces transcription from the promoter.

Question 9

Anti-terminator

Answer 9

Blocks the effect of a transcriptional terminator (by

interacting with RNA polymerases).

Question 10

Lytic versus lysogenic cycle

Answer 10

A temperate bacteriophage has both lytic and lysogenic cycles. In the lytic cycle, the phage replicates and lyses the host cell. In the lysogenic cycle, phage DNA is incorporated into the host genome, where it is passed on to subsequent generations.

Question 11

cro protein

Answer 11

Reduces CII protein synthesis and regulator of cI, since cII activates transcription of cI. Encodes a regulator and represses the cI gene - promotes lytic growth.

Question 12

N protein

Answer 12

Anti-terminator of TN and Tcro (i.e. transcription of N and cro) - early genes.

Question 13

cI

Answer 13

A gene that encodes lambda repressor, which maintains lysogeny.

Question 14

What are the key factors to determine life cycles, i.e. the genetic switch to determine lytic or lysogenic cycles will occur?

Answer 14

cro and cI (lambda repressor) - oppose each other.

Question 15

Transcription terminator

Answer 15

A section of nucleic acid sequence that marks the end of a gene or operon in genomic DNA during transcription.

Question 16

Two types of bacteriophages used in lab experiments? What sort of genetic material do they contain?

Answer 16

T4 and lambda. Both contain DNA.

Question 17

What is a plaque assay?

Answer 17

• A mixture of phages and bacteria is plated on a solid medium. The bacteria grow and produce a bacterial “lawn”.
• The lawn shows areas of clearing where viruses have infected the bacterium and burst their cells – “plaque”.
• The progeny viruses infect and kill neighbouring bacterial cells – producing large plaques or clearing the lawn.

Question 18

What is a prophage?

Answer 18

The genetic material of a bacteriophage, incorporated into the genome of a bacterium and able to produce phages if specifically activated. A prophage is either integrated into the host bacteria’s chromosome or more rarely exists as a stable plasmid within the host cell.

Question 19

What is a lysogen?

Answer 19

A bacterial cell in which a phage exists as DNA in its dormant state (prophage).

Question 20

What are temperate phages?

Answer 20

Phages that have a choice between lytic and

lysogenic pathways, eg. lambda

Question 21

What does the genetic switch refer to?

Answer 21

The trigger for whether the lambda DNA will stay in the lytic cycle, go from the lytic to the lysogenic cycle, or go from the lysogenic cycle back to the lytic.

• If resources are abundant and there are enough nutrients to make many copies of the virus – LYTIC CYCLE
• If resources are limited or marginal – LYSOGENIC CYCLE
• Follows a distinct program of gene expression that is regulated
• Involves a complex genetic switch comprising of several DNA binding regulatory proteins and a set of operators.

Question 22

O and P as gene products of lambda

Answer 22

Initiation of lambda DNA replication.

Question 23

Q as a gene product of lambda

Answer 23

Transcription antiterminator protein for late genes (positive regulator), i.e. allows transcription or mRNA of late genes, such as S, R, RZ, head proteins, and tail proteins (phage structural proteins, or lysis proteins) - one promoter, many genes, i.e. operon. These proteins
• produce the viral structures
• pack lambda DNA, and
• lyse the host cell

Question 24

cII

Answer 24

Activator of transcription of cI (lambda repressor) and int. (integrase). Activate the PRE and PI promoters - cI and int proteins produced. Integrase promotes integration of phage DNA (lysogeny).

Question 25

cIII

Answer 25

Stabiliser of cII.

Question 26

Integrase (int)

Answer 26

Required for site specific recombination with chromosome, as seen in specialised transduction.

Question 27

Relationship between cI and cro (two key lambda genes and their functions).

Answer 27

• When cI is on, cro is off - promotes lysogeny
• When cro is on, cI os off - promotes lytic growth
• Are in competition. Whichever repressor prevails, determines the state of the genetic switch, and of the expression of the lambda genome.
• Two proteins compete to bind OL and OR to decrease synthesis of proteins favouring the counter-pathway.

Question 28

What are the two major promoters for the phage lambda genome?

Answer 28

PL (early promoter for the left operon - goes left), and PR (early promoter for the right operon - goes right).

Question 29

What are the promoters PRE and PRM for?

Answer 29

PRE: transcription for repressor establishment.
PRM: transcription for repressor maintenance.

Question 30

What are the operators OL and OR?

Answer 30

They are the operators that overlap with PL and PR.

Question 31

What does the lambda repressor (cI) act upon?

Answer 31

It binds to OL and OR, which are the operators overlapping with the promoters PL and PR, which inhibits the transcription / mRNA synthesis of the lambda operons, including the genes for the N and cro proteins. It also acts upon the PRM promoter, which activates the production of itself (cI). So it is both an inhibitor and an activator. Promoter of lysogeny. The N and cro proteins (synthesised when the cell was first infected) are unstable and degrade, and their concentrations drop.

Question 32

When conditions favour lytic cycle, which proteins are now produced?

Answer 32

• When conditions favor lytic cycle (eg. nutrient rich so more protease activities which degrade CII) Q and Cro proteins are produced.
• Cro protein dominates & binds to operators OL
  and OR; cI mRNA synthesis inhibited.
• Rightward expression for Q protein to accumulate for Lytic pathway.
• Q protein acts on PR’ enhancing late gene expression.
• Remember that an mRNA that codes many proteins is called polycistronic.

Question 33

What is the molecular anatomy of the OL and OR operators?

Answer 33

They contain three regions - OR1, OR2, and OR3.
* 3 operator sites Overlap two opposing promoters – PR and PRM
* 3 operator sites similar but not identical in sequence
* Cro & lambda repressor cI can bind to any of the three sites – but with different affinity.
When CI is dominant:
* The lambda repressor binds to OR1 and OR2 with high affinity
* The occupation of OR1 and OR2 by the lambda repressor blocks transcription from PR (promoter for the right operon), blocking the transcription of genes for the lytic cycle
* But the transcription from PRM (promoter for repressor maintenance - goes left) takes place (since OR3 is unoccupied), so more cI produced.
When Cro is dominant:
* The Cro repressor binds to OR3 with the highest affinity
* The occupation of OR3 by the cro repressor blocks transcription from PRM, which blocks maintenance of cI transcription but enhances cro transcription from PR, which allows expression of genes for the lytic cycle.

Question 34

What happens when the host DNA, along with the integrated viral DNA, is getting damaged, for exmple, by UV light?

Answer 34

A protein called RecA (a DNA recombination protein) is activated, which leads to the dominance of cro, which causes a switch from lysogenic to lytic pathway.
- The activated RecA cleaves the lambda repressor, rendering it inactive, i.e. literally chopped into two pieces so it can’t bind anymore.