Lecture 9

Question 1

T7 family

Answer 1

Podoviridae

Question 2

T7 capsid and diameter

Answer 2

Naked icosahedral capsid and 60nm

Question 3

T7 unique structure traits

Answer 3

Short tail and tail fibres to grasp surface of bacteria

Question 4

T7 cycle: lytic or lysogenic?

Answer 4

Lytic, no lysogeny

Question 5

T7 genome

Answer 5

Linear dsDNA genome, 40kbp, 56 genes, 59 proteins, 150 bp terminal repeats (identical important for replication)

Question 6

T7 receptor

Answer 6

LPS

Question 7

T7 genome organization

Answer 7

Three classes for the three dif pathways (operons): Class I (early genes, only 3 genes, Dif start sites, same terminator), Class II (more genes, Dif start site, same end site, DNA replication/DNA metabolism, Class III (mature construction of the phage, virion structure and assembly, some start sites in class II, dif end sites)

Question 8

Describe T7 entry into bacterium

Answer 8

T7 tail fibers bind to LPS. The internal core proteins gp14, 15 and 16 can form a channel through the envelope (OM/PG/IM) through which DNA is injected. 850 bp DNA enters the cytoplasm with the assistance of gp16. The E coli RNA polymerase will recognize and bind to the promoter sequence and start transcription. Class I genes are expressed and the rest of the DNA can enter at 40bp/sec. So, entry is coupled to transcription.

Question 9

What are the three early genes?

Answer 9

Gene 0.3: inhibits host type I restriction endonuclease which cleave phage DNA since it is not methylated like host DNA is
Gene 0.7: inhibits E coli RNA polymerase (no more transcription of host genes, full sequestration of necessary machinery)
Gene 1: T7 RNA polymerase unlike phiX174 who only uses host RNA polymerase

Question 10

What are the class II genes and how are they expressed?

Answer 10

• Transcribed by T7 RNA pol (DNA entry at 250bp/sec, much faster than host RNA pol)
• 1.3: DNA ligase, join lagging strand DNA / 2: inhibit E.Coli RNA pol / 3: endonuclease, degrades host DNA, cleaves T7 DNA concatermer / 3.5: lysozyme, inhibits T7 RNA pol / 4: RNA primate/helicase / 5: DNA pol / 6: exonuclease, degrades host DNA and then uses the nucleotides to synthesize own DNA and destroy possible host defences (since no more expression of genes without genome)

Question 11

What are the class III genes and how are they expressed?

Answer 11

• Transcribed by T7 RNA pol once more genome has managed to enter the bacterium
• 8: portal protein / 9: scaffolding protein / 10: capsid protein / 11: tail protein

Question 12

T7 RNA polymerase rundown

Answer 12

• E.coli RNA pol can transcribe from class I promoters but not class II or III, DNA entry proceeds until class II promoters are reached and then T7 RNA pol takes over for entry and transcription
• It was used in vitro to produce SARS-CoV2 since highly processive and can also pair with reporter genes to observe synthesis and stuff
• One of the class I genes encodes T7 RNA pol (monomeric, 100kDa)
• T7 RNA polymerase transcribes class II and III
• A lot of the bigger viruses will carry their own polymerases, because they WANT to be self-sufficient baddies

Question 13

T7 DNA replication initiation

Answer 13

Obvi, need primate/helicase with T7 RNA pol to make primer, then T7 DNA pol can replicate. It always starts at the origin of replication, which is an A-T rich region (less hydrogen bonds to break, lower melting point). RNA pol is at class II promoters (1.1A and 1.1B) and starts transcribing and sometimes this is recognized as primer and replication may happen instead/as well.

Question 14

Humans use telomeres with telomerase (RT), what is the equivalent for T7 phage?

Answer 14

• DNA polymerase needs a primer to initiate DNA synthesis, so the RNA primer at 3’ end of template DNA cannot be removed or replaced with DNA resulting in a loss of sequence, so T7 makes concatemers.

Question 15

How do concatemers form?

Answer 15

The genome has identical terminal repeats, so when the two strands separate and we have new genome copies. The ends can base pair with one another, serving as a primer, so the rest of the genome (where primer was) can be replicated, no sequence is lost. This is a concatemer, multiple copies of the genome stuck together. Then, we get single copies thanks to specific endonuclease that cleave at the terminal repeat sites.

Question 16

Give me a closing statement on T7 DNA replication.

Answer 16

• Bidirectional
• Resembles E.coli synthesis with a continuous leading strand and a discontinuous lagging strand
• T7 encodes all of its own DNA replication proteins
• Unique Origin 5.9kb from left end of genome inhibiting RNA polymerase due DNA replication fork collisions
• DNA replication is initiated by T7 RNA polymerase
• Large concatemers of T7 genomes are formed Bia 150bp terminal repeats

Question 17

Without necessarily any grave details, how does the virus assemble?

Answer 17

The probed assembles and pairs with one of the genome copies derived from the concatemer (encapsidation). Then the probed is matured by adding the tail and fibres. Lysis phage proteins will then create holes in the cell wall to mediate release of the infectious particles. Other proteins are degrading the bacterial genome as well.

Question 18

Lambda phage family?

Answer 18

Siphoviridae

Question 19

Lambda genome?

Answer 19

40-60 kbp, dsDNA, ssDNA extension (cohesive ends), very flexible and has been used to create cDNA libraries

Question 20

Lamda cycle: lytic or lysogenic? Fun fact?

Answer 20

temperate bacteriophage => Lysogenic, has prophage virus, 8% of our genome is retroviral DNA and only about 1-2% encodes protein, are we humans or are we viruses

Question 21

Lambda capsid structure?

Answer 21

• naked Icosahedral head, 63 nm diameter
• Tube like tail, 135 nm long, non-contractile

Question 22

Lambda receptor?

Answer 22

Sugar transports proteins LamB (maltose transporter)

Question 23

What is a temperate phage?

Answer 23

able to grow lytically or exist as a repressed prophage

Question 24

Prophage definition

Answer 24

Integrated phage DNA, lytic genes are repressed, passively replicate with host chromosome

Question 25

Lysogen definition

Answer 25

Bacteria carrying a prophage

Question 26

How does lysogenization work?

Answer 26

Lytic genes are repressed. The prophage is passed down through the cell divisions. Under certain conditions, the lytic genes can be induced and activated to reintegrate he lytic cycle instead.

Question 27

Humans use telomeres with telomerase (RT), what is the equivalent for lambda phage?

Answer 27

The cohesive ends allow for circularization of the genome, so there can be replication with no loss of information.

Question 28

What is the organization of the lambda genome?

Answer 28

Clusters: 5' structural genes, recombination (sib transcription termination site and att attachment site, for integration in E. coli DNA are somewhere in there), regulation (lots of regulation here and this is where lambda phage genes start to express), replication (ori DNA replication origin somewhere in there), late regulation, lysis 3'

Question 29

How is transcription regulated?

Answer 29

At transcription initiation: activator/repressor At transcription termination: antiterminator

Question 30

Describe the transcription events after lambda phage infection (refer to slide 20 of lecture 9)

Answer 30

Two promoters, PL (transcription to the left) and PR (transcription to the right), N is left, Cro is right. N can bind to an RNA sequence (NUTL and NUTR (N utilization site)) that can bind to RNA polymerase to bypass the terminators TL1 and TR1. This allows for the transcription of Q which allows to bypass terminator TR' which results in the expression of proteins involved in DNA replication, lysis, head and tail structure, etc. So if Q is there, the page proceeds to lytic phase. However, there is a protein called Cro right? This is a repressor that binds to operators (OR3, OR2, OR1). There is also another promoter PRM which influences cI transcription. Cro has the highest affinity for OR3, close to PRM (repression maintenance) and shuts down cI. When lots of Cro, starts binding to OR2/OR1 shutting down PR, so shutting down Cro. Lytic or lysogenic depends on the level of cI (suppresses PL and PR). Since cro has highest affinity for OR3, cI binds to OR2/OR1, shutting down PR, so no lytic cycle. Then when cI is high will self-regulate by binding to OR3 as well. cI will form dimers and tetramers and bind to the operators for PR and PL, making a repressor loop and inhibiting the transcription of all other lambda phage genes.

Question 31

How does the lysogenic phase of infection get established?

Answer 31

Well when N suppresses the terminators, CII and CIII are expressed and form a heterodimer that binds to PRE (repression establishment) which activates cI expression. They also bind to PInt which activates integrase expression to integrate phage in genome. They also bind to PAQ (anti Q), shutting down Q, so shutting down lytic cycle. The cII is inactivated by a host cell protease and has a very short half-life (much less stable in metabolically active cells), so it is stabilized by cIII who can inactivate host cell proteases. cI is expressed from PRM when in prophage and makes it stay in prophage.

Question 32

All in all, what determines whether lambda is lytic or lysogenic?

Answer 32

Upon infection, N and Cro are expressed. Cro suppresses cI from PRM. N leads the expression of early genes cII and cIII. In metabolically active cells, cII is unstable, unable to activate cI expression from PRE, so go lytic. In metabolically inactive cells, cII is more stable and reaches high enough concentration to activate cI expression from PRE, so go lysogenic.

Question 33

Let's say the conditions are favourable for the lysogenic cycle, how is the phage genome insertion in the bacterial genome regulated?

Answer 33

If we go lytic, PL will activate transcription until sib, the transcription termination site, which is recognized by RNAseIII which degrades it and the attP site as well as the integrase site, so there is no integration. However, if cII/cIII bind to Pint instead, then sib is not transcribed and there is no recognition by RNAseIII, so the att and int can be expressed and we have production of prophage.

Question 34

How does the phage genome get inserted? (mechanism)

Answer 34

The integrase and host factor are needed to mediate the insertion of the phage genome. An Xis is also needed when the phage genome is excised.

Question 35

When and how does the prophage reintegrate the lytic cycle?

Answer 35

Stresses like UV irradiation can cause DNA damage, activating RecA protein which in turn binds to cI, stimulating the auto protease activity of cI and leads to cI cleavage. Then PL can allow for the transcription of Xis and Int, and with IHF can excise and liberate prophage DNA.