Lecture 19- Bacteriophage Lambda III

Question 1

When does int need to be made?

Answer 1

after infection

Question 2

when do int AND xis both need to be made?

Answer 2

if lysogenic path chosen, for induction

Question 3

What does the repressor protein do?

Answer 3

Produced by the C1 gene, represses all genes in lambda except its own

Question 4

What are the two potential life cycle outcomes from infection?

Answer 4

lytic cycle or cells become lysogens

Question 5

When does the decision get made?

Answer 5

Not at he beginning- delay period of 20 minutes before decision is made

Question 6

What controls the outcome?

Answer 6

level s of cAMP help decide- high in starved cells; low in cells growing in rich medium
high cAMP favors lysogenic response (2nd messenger but also regulatory component)
also, levels of RNase III help decide. high in rich medium, low in starved cells. high RNase III favors lytic response

Question 7

Productive response

Answer 7

Favored in rich medium
cAMP low
RNase III high

Question 8

Non productive response

Answer 8

favored when cells starved
cAMP high
RNase III low

Question 9

Is the outcome ever 100% one or the other

Answer 9

NO

Question 10

What happens during the 10 minute delay?

Answer 10

Infected cell is preparing itself for both outcomes

circular lambda dna is replicating in theta mode; accumulating a lot of dna, prepares for rolling circle replication in case lytic cycle is chosen, or it will have a lot of dna to drive the process of integrating dna

integrase is also being made, only used if cell chooses lysogenic state

reg proteins made too. decision is made: race between making 2 repressor proteins, whichever wins determines outcome

Question 11

Schedule of events in a lambda lytic infection

Answer 11

IE: certain transcription program
DE: delayed early time period has switch to different transcriptional period
L: if decision is to go to lytic cycle, then late transcription happens (lysis will occur)

Question 12

C3 and C2

Answer 12

genes involved intimately in regulation

Question 13

Terminators:

Answer 13

tL termination for transcription at pL
tr1 2 and 3
pr’ has term tr’

Question 14

N

Answer 14

anti terminator function
operates at tL and tr1 and tr2
make mynas that make O and P and Q (another reg protein)
switches the transcriptional program to immediate early to DE period.
transcription at PL in absence of N term at tl
in the presence off N, continues leftward past tL
to right, does not term at tr1 or tr2, term at tr3 instead
key: N acts at two sites, acts on RNA, just at the point where rna pol has transcribed that site in the dna. modifies rna pol s o it no longer terminates

Question 15

C1

Answer 15

codes for repressor

Question 16

PL

Answer 16

directs transcription to left

Question 17

PR

Answer 17

directs transcription to right

Question 18

OL and OR

Answer 18

promoters bound to PL and PR
control two other promoters
?? double check

Question 19

Pint

Answer 19

directs transcription to the left and produces mrna that can be translated to produce int protein gene
p int located in the middle of the xis gene- important

Question 20

PR’

Answer 20

rightward directing promoter. when active, directs to right. It’s the promoter for late genes, goes around circle and gets late genes

Question 21

Immediate early time period

Answer 21

No phage made
pr mostly terminates at tr1, but sometimes at tr2
2 other promoters active: pr’ active, terminates at tr’. Very short, no protein produced. abortive synth of mRNA
a little transcription beginning at p int to give int. most transcription here: PL to produce N protein and PR term at tr1, mostly to produce cro protein

Question 22

CRO

Answer 22

repressor that locks the cell into the lytic cycle/transcriptional program

Question 23

Nut L

Answer 23

N protein and nus complex of proteins act at Nut sites on nascent RNA to modify RNA pol and prevent transcription termination
in the presence of N, transcription machinery (rna pol) passes site called Nut L (n utilization- left of PL)
same one for R
RNA produced has sequence corresponding to NutL
nascent chain will interact with N + nus complex, changes where it terminates
key: N acts at two sites, acts on RNA, just at the point where rna pol has transcribed that site in the dna. modifies rna pol s o it no longer terminates

Question 24

test thinking: transcription beginning at pr’ and term at tr’, now anti termination at tr’?

Answer 24

No. Key is there is no nut site to the right of pr’. if nut site there, you’d get anti termination

Question 25

Q protein

Answer 25

switch from delayed early period to the late period involves the Q protein
accumulating in DE time period
Q mediates the switch to late transcription
also has anti termination function, but works differently than N
Q binds at qut site (q utilization) on DNA. rna pol that begins transcription at pr’ in presence of q would encounter q on dna. rna modified so it would no longer terminate at tr’
it would continue past tr’ and all the way around to include late proteins. both n and q work through anti term mechanism

Question 26

What is Cro protein doing?

Answer 26

Transcription begins at PR, associated operator called OR bound, low level of transcription maintained by repressor that binds to OR (Which is cro)

Question 27

Cro regulation?

Answer 27

Low cro concentrations, transcript that begins at pr is made.
High cro concentrations, cro binds to OR and OL, represses transcription from PR and POL.
Part of what locks cell into lytic cycle
Cro is actually repressing transcription of its own gene. Auto regulation on its own gene
Cro protein relatively unstable. when cro concentration low, get gene being transcribed and produce cro. when high, turns off. When it gets degraded and conc gets low again, turns back on. Like a thermostat (cyclic control mech)

Question 28

Auto regulation of PR by Cro

Answer 28

Cro is the lytic repressor. It also blocks C1 transcription
Low cro concentration, cro is made
high cro concentration, cro bound to OR and OL and not made

Question 29

Int and xis

Answer 29

After infection, only int made. After induction of lysogen, both int and xis proteins made. After infection, only integrase is required. After induction both are required.

Question 30

Regulation of int and xis: infection vs induction

Answer 30

att site is where dna is broken during integration
during delayed early time period, transcription occurs from p int and translates integrase
after infection, transcription begins at PL and proceeds leftward. Initially, mRNA could produce int and xis
when mrna induces sib site, that creates a site for an enzyme called rNase III (high in actively growing cells). this enzyme cleaves RNA after it has crossed the sib site. then rna degraded starting at that site going the right. a little left, probably including N coding region. Prevents rna from producing int and xis proteins
this is why it is after infection that only int is made
After induction of a lysogen:
transcription still begins at pint
tint is no longer there because it does not get integrated. transcription at pint continues on, int still translated, must be a term site eventually
transcription beginning at PL never encounters sib site b/c located beyond att site. this transcription just goes into gal coding region.
transcription beginning at pl in lysogenic configuration is never degraded b/c never crosses sib site
explains why after induction of a lysogen, you make both. xis for excision of prophage dna after induction
sib site IS in prophage genome, just at the other end

Question 31

Delayed early requires

Answer 31

N

Question 32

Late transcription requires

Answer 32

Q

Question 33

Low level transcription produces

Answer 33

Cro and O and P at late times

Question 34

Int made

Answer 34

after infection

Question 35

Int and xis made

Answer 35

after induction of a lysogen

Question 36

C1 repressor

Answer 36

shuts off all the phage genes in the lysogenic state except its own gene

Question 37

Three OR sites

Answer 37

OR1 overlaps PR
OR3 overlaps PRM
C1 binds as a dimer. has highest affinity for OR1. During lysogenic state, dimer binds to OR1, repressing Cro and other genes. OR3 has the lowest affinity
In lysogenic state, as C1 protein made and conc gets higher, it’ll always block OR1 and PR transcription
PR and PL flank C1 gene
Promoter RM = repressor maintenance
transcription from PRM goes to the left, produces mrna that is translated to produce C1 protein
when you have a lysogen, only transcript and protein produced
represses transcription to right at OR and left at OL
C1 concentration gets high, binds OR3 and blocks own transcription from PRM. C1 does same thing as ro. work by reciprocal mechanisms. Cro binds with highest affinity to OR3 and blocks C1 (in lytic cycle). Binds with low affinity to OR1. When bound to OR1 auto reg own transcription from PR. How cro locks into the lytic cycle
Cro prevents repressor from being made- needed for lysogeny
C1 protein must be bound at OR1 and 2 for transcription to initiate at prm. But need transcription from PRM to make C1 protein. How does this work?

Question 38

Which protein is most important for the decision?

Answer 38

C2 protein
Decision is a race between making C1 repressor and cro repressor. C1 wins: lysogen. Cro wins: lytic cycle.
Most important protein for determining the outcome is C2. This is the protein that promotes lysogeny. IT does it as a transcriptional activator.
3 promoters where C2 acts to enhance transcription
-p int
-pre (promoter) (repressor establishment) (rm= repressor maintenance, requires C1)
Other way to transcribe C1: repressor establishment, activated by C2
C2 activates PRE, mrna made and translated to produce C1, which binds to OR site and activates promoter for the repressor maintenance.
third: activates PaQ. directs transcription to left of small RNA which opposes transcription of Q gene. C2 reduces amount of mrna that could be translated to produce Q protein (which promotes lytic cycle)
C2 favoring lysogeny

Question 39

Summary of lambda control circuits

Answer 39

High C2: C1 will win. Low C2: Cro will win
What determines level of C2?
High cAMP blocks C2 degradation by cell protease
C3 also blocks degradation (works in conjugation with C2 to promote lysogeny)
C2 promotes aQ rna, blocks q, then favors lysogeny.
rich medium: high rnase 3. N protein down reg own synth. high rnase III blocks that, more N, get more Q, favors lysis
C1 repressor favors lysogeny
q favors lytic cycle
C1 repressor auto stimulatory and auto regulatory
cro repressor will repress own synthesis