Transcript in Power-Point Format

Chapter 8: Major Shifts
in Bacterial Transcription
Student learning outcomes:
Describe some major shifts:
alternate sigma factors,
new RNA Polymerases,
Phage T4 DNA
multiple promoters
competition between DNA binding proteins
Important Figs: 1*, 2*, 4, 5, 8, 9*, 10*, 11, 12, 13*,14, 17, 18*,
19*, 22*, 23, 24, 26*, 27*
Review Q: 1, 2, 3, 4, 5, 7, 8, 9, 12, 13, 15, 16, 17, 18
AQ: 1, 2, 3, 4, 5, 6, 7
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8.1 Sigma Factor Switching
Phage T4
• Phage infection of bacterium subverts
host transcription machinery
• Establishes time-dependent,
or temporal, program of transcription
– First early phage genes are transcribed (host RNAP)
– Followed by later phage genes
– Late in infectious cycle, no transcription of host genes,
only phage genes
• Change in what genes are transcribed is caused by
a change in transcription machinery:

(s), or new RNAP
8-2
Phage Infection and new s factors
• Recall s is key factor in determining specificity of
early T4 DNA transcription by E. coli RNAP
• To shift transcription to other promoters, s is likely
candidate
• Variant s binds different promoters,
changes specificity of RNAP
• Study of process first defined
in Bacillus subtilis phage, SPO1
Phage SPO1
8-3
Temporal Control of
Transcription
• SPO1 has large genome
• Temporal transcription
program involves new s:
– 1st 5 minutes: expression of
early genes by host RNAP(s43)
– During 5 – 10 minutes:
expression of middle genes
requires phage gp28 s
– After 10 minutes to end: late
genes expressed using phage s
gp33 and gp34
Fig. 1
8-4
Genetic and biochemical
evidence for new s:
• B. subtilis normal RNAP (enzyme
A) has d subunit (helps s)
• Mutations in gp28 prevent early> late switch
• Purify RNAP from cells after
infection; test activity and
promoter specificity
• During infection, phage SPO1
changes promoter specificity of
host core RNAP (b, b’, a)
Fig. 8-5
2
Sporulation occurs in Bacillus
• Change of s mechanism applies
to changes in gene expression
during sporulation:
 sA does vegetative promoters
• Bacteria exist indefinitely in
vegetative state if nutrients are
available
• Under starvation conditions, B.
subtilis forms endospores,
tough dormant bodies
– New sets of genes expressed
Fig. 3
8-6
Sporulation Switching
• Whole new set of genes turned on;
vegetative genes turned off
• Switch occurs largely at level of
transcription (Figs. 4, 5)
• Several new s-factors displace
vegetative s-factor from RNAP core:
s F s E s H…
• Each s-factor has its own preferred
promoter sequence
Lane 1, veg;
Lane 2, sE
Fig. 4: plasmid p213 with two promoters, RNA formed in vitro hybridized to 8-7
Southern blot with these EcoRI-HincII fragments (Fig. 5)
Some Genes have Multiple Promoters
• Some genes must be expressed during 2 or more
phases when different s-factors predominate
• These genes have 2 different promoters
– Each promoter recognized by one of 2 different s-factors
– Ensures expression no matter which factor is present
– Permits differential control under different conditions
Fig. 8
8-8
Bacterial Heat Shock
• Heat shock response: defense to minimize damage:
induces heat shock genes
• Encode Molecular chaperone proteins:
– Chaperones bind proteins partially unfolded by heat
– Help these proteins refold properly
•
Heat shock from alternative s-factor, s32 or sH
– Directs RNAP to heat shock gene promoters
– Accumulation of sH at high temperature due to:
• Stabilization of sH
• Enhanced translation of the mRNA encoding sH
• Responses to low nitrogen and starvation stress also
depend on genes recognized by other s-factors 8-9
8.2 RNA Polymerase Encoded in Phage T7
• Phages like T7, T3, and 11 have small genomes
• 3 phases of transcription: classes I, II, and III
• gene 1 is necessary for class II and class III genes:
– Class 1 genes transcribed by host RNAP
– Gene 1 codes for a phage-specific single subunit RNAP:
Very specific for promoters of class II, III genes
8-10
Temporal Control of
T7 Transcription
• Host RNAP transcribes
class I genes
• One of class I genes is
phage polymerase
• Phage polymerase then
transcribes the class II
and III genes
• [T7 pol widely used: specific
transcription from plasmids
such as pUC18 vitro or vivo]
Fig. 9 8-11
8.3 Infection of E. coli
by Phage lambda l
• Virulent phage (like T4, T7) replicate, kill their host
by lysing or breaking it open
• Temperate phage, such as l, infect cells but don’t
necessarily kill
• Temperate phage have 2 paths of reproduction:
– Lytic: infection progresses as in a virulent phage
– Lysogenic: phage DNA integrated into host genome
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Two Paths of Phage Reproduction
Fig. 10
8-13
Lysogenic Mode
Lytic Reproduction
• Phage protein (l repressor, CI) • Phage l has 3 phases of
made, binds 2 operator regions
transcription:
• CI shuts down transcription of
– Immediate early
all genes except for cI gene
• (host RNAP)
– Delayed early
• (needs Cro, N)
• Lysogen: bacterium harboring
integrated phage DNA
– Late
• (needs Q)
• Prophage: integrated DNA
• Genes arranged
sequentially on phage DNA
Outcome depends on protein-DNA interactions;
protein-protein competition
8-14
Genetic Map of Phage l
• Linear DNA in
phage particle
• After infection,
DNA circularizes
using 12-bp
sticky ends
(cohesive (cos)
ends)
• Initial
transcription uses
host RNAP
Fig. 11
8-15
Lytic infection uses
antitermination of transcription
• Host RNAP transcribes IE genes;
rho-dependent terminators (t).
•
Antiterminator gene product N
permits RNAP to ignore terminators,
continue to DE transciption
• Late genes are transcribed from L
promoter when another Q protein
antiterminator permits transcription
to continue without premature
termination
Fig. 12
8-16
Lytic Infection uses
Antitermination of Transcription
and repression of repressor
One of 2 IE genes was N:
– Used for antitermination
Other IE genes is cro
– cro encodes repressor of cI
gene that allows lytic cycle to
continue
8-17
N Antitermination Function
• Sites surrounding N gene:
– Left promoter, PL
– Operator, OL
– Transcription terminator (t)
• When N is present:
– N binds transcript of N
utilization site (nut site)
– Interacts with protein complex
(NusA, B) bound to RNAP
– RNAP ignores transcription
terminator, continues into
delayed early genes
Fig.8-18
13
Lambda Lytic:
Antitermination of Late
region requires Q
• Q binds to Q-binding region of DNA (qut site) as
RNAP is stalled just downstream of late promoter
• Binding of Q to RNAP appears to alter enzyme so it
ignores terminator and transcribes late genes
Fig. 17
8-19
Establishing Lysogeny shuts off Lytic Genes
• Phage establish lysogeny by:
– Production of repressor CI to bind to early operators
– Preventing further early RNA synthesis
• DE transcription from PR produces cII mRNA translated to CII
• CII allows RNAP to bind PRE and transcribe cI gene, resulting
in repressor
8-20
Autoregulation of
cI Gene Maintains
Lysogeny
l repressor CI binds as dimer
to l operators, OR and OL
CI turns off further early transcription:
• Interrupts lytic cycle
• Turnoff of cro very important: Cro
acts to counter CI activity
Stimulates own synthesis by activating PRM
– 3 operator sequences permit
cooperative binding of CI
Fig. 19
8-21
Lambda Repressor
Protein
cI protein – 27 kD
Dimer of 2 identical subunits:
Each has 2 domains:
• NH2-terminal DNA-binding
• COOH-terminal is site of
repressor-repressor
interaction for dimerization
and cooperative binding
• No inducer
Fig. 9.5 CI binding
operator OL2
8-22
Involvement of OL in Repression of PR and PRM
• Repressor binds to OR1
and OR2 cooperatively,
but leaves OR3
• RNAP binds to PRM,
overlaps OR3 to contact
repressor bound to OR2
• Protein-protein
interaction required for
PRM promoter to work
efficiently
• High levels of repressor
can repress
transcription from PRM
Fig. 22
8-23
Evidence for RNAP: CI
direct interaction from
Intergenic Suppression
• Mutation in one gene suppresses
mutation in another
• Mutant with compensating amino acid
change in RNAP subunit s restored
interaction with mutant repressor
• Direct interaction between CI
and RNAP is necessary for
efficient transcription from PRM
Fig. 23
Fig. 25; CI contacts s
8-24
Fate of l Infection
• Delicate balance lysis: lysogeny
• need DE expression
• Phage particles on bacterial lawn
– If lytic infection occurs
• Progeny spread ,infect other cells
• Circular hole in lawn is plaque
– Infection 100% lytic clear plaques
– Plaques of l are usually turbid
meaning lysogen is present
(resistant to infection)
• Some infected cells get lytic cycle,
others are lysogenized
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Battle Between cI and cro determines fate
of lambda infection
• Lysogeny: cI gene codes for
repressor, blocks OR1, OR2,
OL1, and OL2;
– turns off early transcription
(including cro)
• lytic infection: Cro gene
codes for Cro, blocks OR3 and
OL3,
– turns off transcription of cI
• Gene product first in higher
concentration determines fate:
– cII important here
Fig. 26
8-26
Lysogen Induction
• When lysogen suffers DNA
damage, bacterial SOS response
is induced
• Initial event is coprotease activity
of RecA protein (normally used
for DNA recombination)
• Causes CI to cut itself in half,
removing it from l operators
• Lytic cycle is induced
• Progeny phage escape
potentially lethal damage of host
Fig. 27
8-27
Review questions
4. Summarize mechanism B. subtilis cells use to alter
transcription program during sporulation.
17. Present a model to explain the struggle between cI
and cro for lysogenic or lytic infection of E. coli by l.
7. How does phage T7 control its transcription program?
What is the difference between antitermination (l) and
attenuation (trp operon) in regulating transcription?
8-28