lambda phage

Question 1

explain how transcription and translation occur

Answer 1

transcription occurs via RNA polymerase on the DNA strand to generate mRNA
-translation makes use of the ribosomes and the shine dalgarno sequence ensures the mRNA is correctly aligned onto the ribosome

Question 2

what type of phage undergoes the lytic cycle

Answer 2

virulent phages

Question 3

what type of phage undergoes the lysogenic cycle

Answer 3

a temperate phage

Question 4

what is a prophage and a lysogen

Answer 4

a prophage forms when a phage integrates into the host genome and a lysogen is a bacteria containing a prophage

Question 5

explain the steps of phage cell entry

Answer 5

1. the phage adsorbs to the host cells lamB protein using its J protein
2. then the linear double stranded dna is transported over the membrane via the pstM sugar protein into the host cell

Question 6

how does the linear phage genome circularize

Answer 6

once inside the host; it circularizes using its cos sites and DNA ligase seals the nicks

Question 7

how does the circular phage genome supercoil

Answer 7

it used DNA gyrase to nick the DNA and form supercoils
-the nicks are closed using DNA ligase
-supercoiling allows transcription to occur

Question 8

how does the phage initially replicate

Answer 8

using theta replication via O and P proteins

Question 9

explain the phage genome organisation

Answer 9

there are 73 genes all organised into clusters by function
-this helps with the cascading of regulatory events in the lytic and lysogenic cycle
-it also helps regulate genes by co ordinated transcription from the same promoter

Question 10

what are the 3 phases of phage transcription

Answer 10

1. immediate early
   2.delayed early
2. late lytic or late lysogenic

Question 11

what happens in the immediate early phase

Answer 11

RNA polymerase will transcribe N protein from PL and cro protein from PR

Question 12

what happens in the delayed early phase

Answer 12

then N protein will perform autoregulation where it binds to the boxB sequences found in the mRNA of nutL and nutS. it binds with host nus proteins and RNA polymerase so that RNA polymerase can read through the TI; TL, TR1 and TR2 terminators to transcribe all the other proteins

Question 13

what proteins do we get in delayed early phase

Answer 13

from PL= N, CIII, int and xis
from PR= cro, CII, O, P and Q

Question 14

what is retroregulation at the sib site

Answer 14

now that we have int and xis we need autoregulation to occur at the sib site
-this is where the sib site forms a hairpin loop which is detected by RNASE III to cleave it
-the now free 3 prime end is degraded by exonuclease so that some int is degraded
-leaves more xis than int so that neither occur before the decision has been made

Question 15

CII transcriptional activation

Answer 15

now that we have CII it can bind to PAQ; PRE and PI to get antisense Q mRNA, C1 and int respectively

Question 16

how does cro and CI initiate lysis and lysogeny

Answer 16

cro will bind the OR3 site of OR operator and block transcription from the PRM promoter so no more C1 can be made
-it allows transcription from PR for more cro to be made
-as cro increases it binds the OR2 and OR1 sites aswell so that materials are not wasted and no more cro can be made
-C1 binds OR1 and OR2 to block cro transcription from PR but allows transcription from PRM for more C1

Question 17

what decides whether lysis or lysogeny will occur

Answer 17

decided by the environmental status of the bacterial cell

Question 18

how does the phage gauge the environmental status of the cell

Answer 18

1. FTSH/HFLBCK protease
   -this protease is high when cAMP is low ie; glucose conditions are good
2. RNase III
   -this is produced in response to cell growth
   -so if growth is good then RNase III is high
   -it can increase N protein translation by 200 fold and it can degrade CII mRNA

Question 19

what monitors the health of the cell

Answer 19

CII
- remember CIII protects CII to extend the half life from 1 to 5 minutes
-remember that RNase III degrades CII and FTSH is also capable of degrading CII and CIII

Question 20

explain what happens when the lytic pathway is chosen

Answer 20

in this instance the conditions in the cell are good and so RNase III will degrade CII so no CI is made and thus; in delayed early transcription cro will prevail
-so cro can bind to OR3 and promote more cro to be made and inhibit C1 from PRM
-transcription from PR allows more O,P and Q protein to be made
-so the O and P proteins can initiate early theta replication and Q protein can accumulate so that in the late lytic phase; Q protein is high enough that even though cro has now bound to OR1 and OR2 aswell; Q protein can still bind to PR dash qut site to read through TR dash terminator so that the lytic proteins can be transcribed

Question 21

what are the 4 requirements for lysogeny

Answer 21

1. monitoring the health of the bacterial cell
2. repression of lytic functions
3. integration of the phage
4. maintaining lysogeny

Question 22

what must the cell conditions be for lysogeny to occur

Answer 22

1. low glucose
   -low FTSH and RNase III
   -so high C11
   -so CI can be made
2. the gene dosage of CII needs to be high enough ie; an MOI of 1 is too low but an MOI of 10 provides sufficient CII for lysogeny to occur

Question 23

1. monitoring the health of the bacterial cell

Answer 23

when the 2 conditions for lysogeny are met; this means that CII prevails and can bind to PAQ, PRE and PI for transcription

Question 24

2. repression of lytic functions

Answer 24

now in late lytic; C1 is made from PRE and can now bind to OR1 and OR2
-this inhibits transcription from PR so no more cro is made and also inhibits transcription from PL so no more N protein is made and thus; no more immediate early transcription so no more cro
-then when C11 binds to PAQ it transcribes an antisense Q mRNA that prevents Q protein from being made
-lastly; the PRE transcript for CI protein overlaps with the cro mRNA transcript from PR and so this PRE transcript is also antisense cro mRNA that binds to cro mRNA and prevents transcription of cro

Question 25

3. phage integration

Answer 25

now the phage is ready to integrate as lysogeny has been chosen and so when CII binds to PI it transcribes integrase -for integration we need more int than xis -also need the help of IHF protein -now via recombination of 2 site specific sequences( attP on the phage and attB on the host genome) the phage integrates -this leads to POP and BOB becoming POB and BOP. -note that sib site has moved away from int and xis

Question 26

4. maintenance of lysogeny

Answer 26

maintenance done solely by C1 and DNA looping 1. remember that CI was produced from PRE -this binds to OR1 and OR2 to allow for transcription from PRM for more C1 -then as C1 rises it binds OR3 aswell; preventing transcription of more C1 -then as C1 drops it unbinds from OR3 first so more C1 can be transcribed from PRM -this negative feedback loop maintains lysogeny -also when CI is bound to OR sites; it undergoes octamerization to form a DNA loop between the 2 OR sites to maintain the lysogenic state in the face of noisy gene expression; non specific CI DNA binding and operator site mutations

Question 27

what is the lysogenic conversion

Answer 27

not only does C1 maintain lysogeny but it also provides immunity to the bacterial cell by preventing superinfection by other phages

Question 28

what are the 5 other genes that are transcribed on the phage once it has integrated

Answer 28

rexA, RexB and sirB -these all provide immunity to the bacterial cell to block infection by other phages lom- this gene provides increased pathogenicity by increasing the bacterial cells ability to bind to mammalian cells Bor-this protects the bacterial cell against serum killing

Question 29

what happens at the Poop promotor

Answer 29

when transcribed it produces an antisense CII mRNA that binds to C11 mRNA so that CII cannot be made -this allows the phage to enter lysis -so in lysogeny this promoter is inhibited by lexA

Question 30

how does induction occur in a non phage infected cell

Answer 30

the DNA damage activates recA which cleaves lexA off of the SOS response genes so that damage repair proteins can be made

Question 31

how does induction occur in a non phage infected cell

Answer 31

the DNA damage activates recA which cleaves lexA off of the SOS response genes so that damage repair proteins can be made

Question 32

how does induction occur in a phage infected cell

Answer 32

1. The DNA damage activates recA which firstly cleaves C1 off of OR1 and OR2 so that PR and PL can be transcribed again -this allows for cro, O, P and Q to be made once more - and N protein so that more int and xis can be transcribed -the cro can now bind to OR3 to block PRM transcription of more CI so lysogeny cannot be re-entered 2. then recA also cleaves lexA off of the Poop promoter so that CII is not made and thus; no more lysogeny

Question 33

how does excision occur

Answer 33

-now that N protein has allowed for the transcription of more int and xis and the sib site has moved away; we now have equal amounts of both for excision to occur -once excision happens; the O and P proteins are used to initiate theta replication -the Q can bind to PAQ and allow for transcription of the late lytic genes