Lesson Notes
Gene Regulation and Development: Phage —Handout 1
-Choose between lytic and lysogenic pathways
-Lytic pathway
-Immediate Early transcription—0-2’ after infection
-Regulatory proteins
-2 promoters—PL and PR
-2 genes transcribed—N and cro
-2 terminators—tL and tR1
-Delayed Early transcription (N-dependent)—2’-5’
-Replication of phage DNA
-Genes transcribed going left from N = cIII, red, xis, int
-Genes transcribed going right from cro = cII, O, P, Q
-Most important genes = O,P
-Needed for DNA synthesis
-N protein allows reading through terminators of Immediate Early
transcription
-Either anti-terminator or activator of unknown promoter placed to
the left of tL and to the right of tR1
-If activation, then IE RNA and DE RNA should be
distinct; or if anti-termination, then DE RNA should be
extension of IE RNA
-If activation, then DE RNA might not depend on PL and
PR; or if anti-termination, then DE RNA depend on
initiation at PL and PR
-Biochemically shown that N is anti-terminator
-If supply N to PR-PL- cell and N is activator, then DE
transcription should occur—NOT WORK
-If supply N to lysogenic cell with cI present and N is
activator, then DE transcription should occur—NOT
WORK
-Late transcription (Q-dependent)—5’-10’
-Make phage components and lyse cell
-PR’
tR’
-Q allows PR’ to transcribe
-Q is necessary for late transcription
-Anti-terminator protein of tR’
-Since DNA is circular, A through J are transcribed
-Code for head and tail proteins of phage particles
-Aspects of lytic pathway
-Transcriptional (regulatory) cascade
-IE
N
Q
late transcripts
-N and Q are anti-terminators which act as positive regulators
-N is a RNA binding protein
-Interacts with RNAP as RNAP passes through nutL and nutR in
mRNA
1
- nutL and nutR are secondary binding within mRNA
-Stops RNAP from recognizing terminators
-TAT protein of HIV like N
-Q recognizes a DNA site which overlaps with PR’
-Interacts with RNAP to read through terminators
-Lysogenic pathway
-Clear plaques = lysogenization mutants
-3 mutants found—cI, cII, and cIII
-Cross streak each and if they complement each other, then turbid plaques
form in intersection of cross
cIcIIcIIIcI
+
+
cII
+
cIII-+ = turbid plaques = complementation
-- = clear plaques = no complementation
-What mutant genes code for
-cI = repressor
-Activates PRM (promoter for repressor maintenance) while
represses PL and PR
-Leftward transcription of cI from PRM
-Initially transcribes leftward from PRE (promoter for repressor
establishment)
-PRE is a weak promoter, therefore needs activator
-cII = DNA binding protein
-Activate PRE transcription of cI
-Activate Pint which codes for integrase
-Easily degraded
-cIII = inhibitor of cII break down
-Stabilize cII
-cII and cIII are delayed early genes
-Both need N
-How does integration occur?
-cII and cIII turn on PRE, which transcribes cI
-Repressor (cI) binds to OR and OL, halting lytic pathway after Immediate
Early transcription
-Halts transcription of N; therefore, halts transcription of cII and
cIII
-PRM continues repressor transcription with positive activation by cI after
PR and PL are repressed
cI
OR3
OR2
OR1
cro
PRM
PR
-Repressor is able to bind to 3 sites, between PRM and PR—OR1, OR2, and
OR3 in that order—Handout 2 Fig, 2.1, 2.2
2
-When bound to OR1, shut down PR by preventing RNAP to bind
-Overlaps –10 region of PRE
-Binds to OR2 at approximately the same time due to stabilization
of binding to DNA through protein-protein interactions—Handout
2 Fig. 4.18
-When bound to OR2, it touches RNAP on PRM
-OR2 is 1 bp closer to PR than to PRM—allows for different
interactions between cI and RNAP
-Acts as negative regulator of PR
-Overlaps 3 bp of –35 region of PR by 1 bp, which
prevents RNAP from binding
-Acts as positive regulator of PRM—Handout 2 Fig. 1.16
-Interacts with -subunit of RNAP at –35 region of
PRM
-Overlaps -35 region by 2 bp
-When bound to OR3, there is too much repressor present and halts
transcription of cI until levels decrease enough so that not bound to
OR3 anymore
-Overlaps with –35 region of PRM
-Allows for reversibility of repression
-Needed for induction of prophage
-Aspects of lysogenic pathway
+
-N
cII, cIII
PRE
cI
PRM
-Regulatory cascade
-cI PRM loop called autogenous regulation
Uses of Recombinant DNA Technology, in general
-Protein overproduction
-Agriculture, drugs (insulin), chemical products
-Vector with high-level production signals just before inserted DNA—overexpression of protein
-Proper transcription and translation initiation signals
-Gene Regulation
-Hard to assay for particular gene
-Would like to change conditions (i.e., add or subtract nutrients, chemicals, etc.)
and see what happens
-Expression vector—i.e., -galactosidase = reporter
-Regulatory sequence of gene of interest attached to reporter gene
-Re-insert into organism of choice and watch for any effects by assaying for
reporter
-DNA sequence analysis
-DNA sequence leads to aa sequence which leads to protein structure
-Can tell what the order of aa does when combine into certain order (distinct
proteins)
3
-Tell DNA function from structure
-Make mutations in test tube
-Replace wild-type with mutant in organism
-“Reverse Genetics”
-Protein-DNA interactions
-Quantify RNA synthesis
-Use DNA (cloned gene) as probe for its mRNA product
-Genome Projects
-So far, 25 bacteria (most are known disease carriers), H. sapiens, D.
melanogaster, C. elegans, S. cerevisiae
-Evolution at molecular level
Use of Recombinant DNA technology to Study Transcriptional Control
-ALL Figures taken from Genetic Switch by Ptashne—Handout 2, 3
-DNAse I Footprinting—Protection Assay
-Radio-label one end of DNA chain
-Treat with DNAse I which cuts anywhere
-Want Mild cut— ~1 cleavage per DNA molecule
-Generate equivalent of DNA sequencing gel
-But combined into 1 lane
-Ladder with every step represented
-If protein bound to DNA, then protect DNA from cleavage where protein bound
-Will see gap in ladder = footprint
-cI—OR interactions using DNAse I Footprinting Assay
-At low cI concentration, see 2 sites with partial cleavage
-OR1 and OR2 partially protected
-At medium cI concentration, see 3 sites
-OR1 and OR2 fully protected
-OR3 partially protected
-At high cI concentration, all 3 sites fully protected
-When line up all 3 OL and OR, 2-fold symmetry almost exists
-Table 2.1, 2.2
-12 out of 17 bases perfectly symmetrical
-Consensus = TATCACCGC
-1st A and C, 12/12 the same between all
-3rd C, 11/12 the same between all
-Down mutations at these 3 sites are strongest
-cI—OR Binding Constant using DNAse I Footprinting Assay—Handout 3A
-Relative amount of cI to get 50% protection
-Cooperative = all 3 at same time
-Intrinsic = each one individually
operator
cooperative intrinsic
wt OR1
1 unit
2 units
wt OR2
1 unit
25 units
wt OR3
25 units
25 units
-cI (dimer) at OR1 and OR2 help each other bind—Handout 3B
4
-Repressors at OR1 helped a little
-Repressors at OR2 helped a lot
-cI (dimer) at OR3 not interact with other cI
operator
cooperative intrinsic
OR1C
----wt OR2
5 unit
25 units
wt OR3
5 units
25 units
-Knock out OR1, then OR2 and OR3 can interact—Handout 3C
-OR2 binding is weaker since no interaction with OR1
(primary interactions)
-OR3 binding is stronger since interactions now available
-Genetic constructs used in reporter assay to show—Handout 2 Fig. 4.19
-On plasmid,
-cI under control of lac operon promoter and operator
-LacI gene also on plasmid
-Construct 1 chromosome,
-OR1-OR2-OR3-PRM-LacZ gene
-No IPTG, then no cI, then PRM OFF
-With IPTG, then cI, then PRM ON
-Construct 2 chromosome,
-OR3-OR2-OR1-PR-LacZ gene
-No IPTG, then no cI, then PR ON
-With IPTG, then cI, then PR OFF
-When vary IPTG, leads to variation of active cI, leads to variation of -galactose
synthesis
-See effects of cI concentration on PR and PRM
-Plot -gal levels as function of IPTG concentration
-Active OR1, OR2, OR3—Handout 2 Table 4.2, 4.3 and figure on page
-PR—-gal levels decreases as cI increases
-PRM—-gal levels increase as OR1 and OR2 filled, and decreases
as OR3 filled
-Around same time that PR shut off, PRM turned on
-Active OR1 with mutant OR2 and OR3—Handout 2 Fig. 4.21 top
-PR—-gal levels decreases as cI increases
-PRM—-gal levels never turned on
-OR1 alone repress PR, not activate PRM
-Active OR3 with mutant OR1 and OR2—Handout 2 Fig. 4.21 middle
-PR—-gal levels never shut off
-PRM—-gal levels never turned on
-OR3 represses PRM, no effect on PR
-Active OR2 with mutant OR1 and OR3—Handout 2 Fig. 4.21 bottom
-PR—-gal levels decrease as cI increases
-PRM—-gal levels increase as cI increases
-OR2 alone activates PRM and represses PR
-Need more repressor for each since no cooperative binding
at OR1 and OR2
5
-Active OR2 and OR3 with mutant OR1—Handout 3D
-PR—-gal levels decrease as cI increases
-PRM—used PRM up-1 mutant–does not need cI to activate
-Looks more like consensus
--gal levels ON when no cI
--gal levels decrease as cI increases
-OR3 represses PRM
-Summary Tables
-xxx = cI dimer bound to site
-ooo = mutated site so no binding of cI can occur
lacZ—PRM
OR3 OR2 OR1 PR—lacZ
OFF ooo
ooo
xxx
OFF
ON
ooo
xxx
ooo
OFF
OFF xxx
ooo
ooo
ON
OFF ---------ON
ON
---xxx
xxx
OFF
OFF xxx
xxx
xxx
OFF
-Last 3 are physiologically relevant
- --- = empty, not mutated
-Same experiments done with cro protein
-cro bind also to OR1, OR2, OR3
-Tightly to OR3, then OR2, then OR1
-No cooperative action
-Low cro, then shut off PRM, therefore shut off cI
-High cro, then shut off PR
6