Module 6: Reg. of Gene Expression (Global Gene Reg.) Flashcards
Global Gene Regulation
Refers to the coordinated and simultaneous regulation of MULTIPLE genes (of different operons)
In what overall instances is global gene regulation needed?
When an environmental change requires altered expression of a greater # of genes that are on different operons
AND
for cell responses that require the simultaneous increase and decrease of expression of different genes
Regulon
Collection of operons (genes + operators) that are globally affected by the SAME regulatory protein
Regulons contain genes that are…
coordinated to respond to the same regulatory systems
Examples of global gene regulation (2)
1) Catabolite repression (glucose preference in E.coli)
2) SOS Response
Catabolite Repression
Process of inhibiting operons that utilize alternative substrates (carbon sources/nutrients) by the presence of a preferred carbon source (usually glucose)
Catabolite repression in E.coli
Glucose presence inhibits pathways producing enzymes that catabolize OTHER nutrients/carbon sources
–> Allows for preference of glucose to be exhibited
CRP-cAMP complex regulates how many genes?
> 100 operons = Constitutes a major regulon!
What is one of the most characterized regulons?
SOS (DNA damage) Response System
SOS Response
One type of DNA repair systems that responds to SERIOUS DNA damage
= A final option for rescuing cells whose genomes have been seriously damaged!
The SOS system is a ______ system used for ________ that is activated upon__________
multi-gene (REGULON) system for wide-scale DNA repair that is ACTIVATED upon detection of DNA damage
Who famously studied DNA repair mechanism in its early stages?
Jean-Jaques Weigle
What did Jean-Jaques Weigle study?
Studied DNA repair via analysis of lambda phage infectivity in E.coli (both of which were treated and untreated with UV!)
What was Jean-Jaques Weigle’s hypothesis?
UV light exposure to E.coli triggers INDUCTION of repair of DNA-damaged phage in a process that also generates mutations
What was Jean-Jaques Weigle’s experiment (process)?
1) Treated lambda phage with UV light
2) Treated ONE group of E.coli with UV light (and the other group was untreated)
3) Infected UV-exposed and NON-UV E.coli cultures with the UV treated phages!
4) Observed production of phage progeny and whether any mutations arose
What were the observations of Jean-Jaques Weigle’s experiment?
UV Treated E.coli = Had > # of progeny produced BUT a > rate of mutation (as seen in the progeny)
NON-UV E.coli = Had < # of progeny produced BUT a < rate of mutation (as seen in the progeny)
= CORRELATION between infectivity and mutagenesis!
What relationship was observed in Jean-Jaques Weigle’s results?
A correlation between infectivity (# of produced progeny) and mutagenesis (# of mutations in the progeny)
What were the conclusions drawn from Jean-Jaques Weigle’s experiment?
The observed correlation suggests that:
1) UV treatment of host cells enhanced their ability to repair UV-induced DNA damage in phage DNA = greater phage replication
2) The DNA repair mechanism is error prone (greater # of mutations in the progeny)
Chloramphenicol
An antibiotic that functions by blocking protein synthesis in bacteria
After Jean-Jaques Weigle’s work, what was used to attempt to further characterize DNA repair mechanisms?
Studies were conducted using chloramphenicol!
In the chloramphenicol studies what was observed?
What was the conclusion? Future questions?
Observed = chloramphenicol treatment resulted in BLOCKED DNA repair!
Conclusion = DNA repair mechanism requires protein synthesis
New Question = Which proteins?
What did experiments with chloramphenicol discover?
Discovered that DNA repair mechanisms must require some amount of protein synthesis
(as DNA repair was shut down when protein synthesis was shut down)
Why was the lacZ promoter-probe transposon first used?
To identify which genes produced DNA repair proteins!
lacZ promoter probe transposon
A transposon with two distinct DNA regions:
1) Reporter Gene (lacZ without promoter)
2) Selection Gene (ampicillin resistance)
Reporter Gene
A promotorLESS gene that is NOT expressed UNLESS it is inserted within an actively transcribed gene!
ln the lacZ promoter probe transposon, lacZ is only expressed if…
The transposon randomly inserts itself into an actively transcribed gene!
What is a main difference and similarity between the reporter gene and selection gene in the lacZ promoter probe transposon?
Difference:
lacZ reporter = DOES NOT have its own promoter (can ONLY be transcribed if inserted into an ACTIVE GENE)
AmpR= HAS its own promoter (can be transcribed if inserted anywhere)
Similarity:
–> BOTH require insertion into the host chromosome in order to be transcribed (otherwise they will be degraded before transcription can occur)
Colonies incubated with lacZ promoter probe transposon:
Plated on ampicillin
Colonies that survive and grow have ampicillin resistance
== Transposon insertion OCCURRED (but does not specify WHERE the insertion happens)
Colonies incubated with lacZ promoter probe transposon:
Plated on X-Gal. (+ ampicillin)
BLUE colonies = Expressing B-galactosidase = lacZ was transcribed == Transposon inserted into ACTIVE gene!
WHITE colonies = NOT expressing B-galactosidase = lacZ was NOT transcribed and therefore the transposon did NOT insert into active gene!
What else was used along with the lacZ promoter probe transposon to identify DNA repair encoding genes?
A DNA damaging agent = MITOMYCIN C
Mitomycin C
A DNA damaging agent
Screening process for mutants with promoter-probe insertion in DNA repair genes:
1) Incubate cells with the lacZ promoter-probe transposon = random insertion
2) Plate cells with AMP, X-GAL, Mitomycin C (to induce DNA damage and trigger DNA repair response)
3) Isolate the BLUE colonies (= colonies with insertion into active genes WHILE DNA damage response is occurring)
4) Replica plate the blue colonies: new plate with AMP, X-GAL., and NO MmC
5) The WHITE colonies on the non MmC plate = probe insertion into genes NOT active in the absence of DNA repair mechanism
6) Isolate these white colonies (they have insertions in genes that encode for DNA repair!)
7) Locate transposon insertion sites
8) Analyze the gene fusions to determine which genes are involved in DNA repair
When using the promoter-probe to isolate DNA repair genes, what colonies did the scientists want to collect?
The colonies that had LacZ expression in the PRESENCE of MmC but NO LacZ expression in the ABSENCE of MmC
= colonies with insertions in DNA repair genes (because DNA repair genes will technically only be active when DNA damage is occurring but be inactive when there is no damage!)
dinGenes
“Damage Induced Genes”
–> Many of which are part of the SOS regulon
What genes were discovered from the use of lacZ promoter probe transposon screening?
Where were they found?
dinGenes!
–> Found ALL OVER the genome and many were found to be part of the SOS regulon
How do bacterial cells detect DNA damage?
By detecting the PRODUCTS of DNA damage = single stranded DNA (ssDNA)
Bacterial cells recognize DNA damage by ________________ and NOT _____________
Bacterial cells recognize DNA damage by detecting ssDNA (product of damage)
and NOT by detecting the cause of DNA damage (damage agent)
What genes of the SOS regulon were found to alter the SOS regulon response?
1) lexA
2) recA
lexA gene
= encodes for the LexA protein which acts as a repressor for operators of the SOS regulon
LexA
A repressor for operators of the SOS regulon
recA gene
= encodes for the RecA protein, an ssDNA binding protein
RecA
an ssDNA binding protein that attaches to single-stranded DNA upon DNA damage
–> triggers the cleavage of LexA
What is the relationship between LexA and RecA?
When RecA is bound to ssDNA, it triggers the cleavage of produced LexA
== Results in dysfunctional form of LexA = Can’t bind to SOS operators and as such, transcription of SOS regulon is initiated!
–> Together, LexA and RecA trigger the initiation of SOS regulon transcription, beginning the SOS response in cells!
SOS regulon consists of how many genes?
> 40 genes dispersed throughout the genome
SOS regulon:
DNA damage present
–> High production of LexA and RecA
1) RecA recognizes and binds to ssDNA at sites of DNA damage
2) RecA in its bound state triggers cleavage of produced LexA
3) Cleaved LexA CANNOT bind operators
4) RNA polymerase binds to free promoters of SOS regulon
5) Transcription of the operons in the SOS regulon begins and the SOS response is initiated
6) Leads to DNA repair!
SOS regulon:
NO DNA damage present
–> Low production of LexA and RecA
1A) RecA is produced but does not bind to DNA
1B) LexA is produced and does NOT get cleaved by RecA = LexA is in its functional state
2) LexA binds to the operators of the SOS regulon
3) RNA polymerase cannot bind to the operators
4) Transcription of operons in the SOS regulon cannot be initiated and NO SOS regulon response occurs
Other than regulons, what is another method of global gene regulation?
Sigma Factors
Sigma Factor
A protein that binds to the core of RNA polymerase which allows for RNA polymerase to:
1) FIND
2) BIND
to specific promoters of specific genes!
The use of different sigma factors causes…
RNA polymerase to be directed to different genes
== Resulting in the transcription of certain genes over others!
What is the most common sigma factor?
Sigma-70 (RpoD)
Sigma-70
AKA RpoD == PRIMARY SIGMA FACTOR
–> The sigma factor responsible for recognizing MOST promoters!
Alternative Sigma Factors
Sigma factors which recognize different promoters BUT are all still related to sigma-70 (primary SF)
Sigm-54
AKA RpoN (think N for nitrogen)
–> A family of sigma factors
–> Involved in regulation nitrogen utilization genes!
Other than sigma-70, most bacteria have 1-2 SFs in this family…
Sigma-54 (RpoN)
How does sigma-54 differ from sigma-70?
Sigma-54:
1) Requires an activating protein AND ATP to initiate transcription (sigma-70 does not)
2) Recognizes promoters at different distances from the initiation of transcription (sigma-70 does not)
Other than sigma-70, what are some common alternate sigma factors?
1) Sigma-54 (RpoN)
2) Sigma-32 (RpoH)
3) Sigma-38 (RpoS)
Sigma-32
AKA RpoH
–> Regulates heat shock genes; usually involved in proper protein folding
–> Recognizes ~30 promoters
Under normal conditions sigma-32 is usually…
How does this differ from high temp conditions?
Normal conditions:
Sigma-32 is quickly degraded by proteases
High temps:
–> Proteases are too “busy” dealing with denatured proteins that sigma-32 can accumulate in the cell and trigger heat-shock response!
Sigma-38
AKA RpoS
–> Regulates general stress response genes
Sigma-38 accumulates in cells in what conditions?
General stress conditions:
1) High cell density
2) Nutrient starvation
3) Low temp
4) High osmolarity
5) Oxidative stress
Common target genes of sigma-38 are involved in…
1) General stress adaptation
2) Protein processing
3) Membrane stability and transport
4) Metabolism
Sigma factors can control…
ENTIRE REGULONS
Sigma factors are regulated themselves by…
ANTI-Sigma Factors
Anti-Sigma Factors
Molecules that bind to sigma factors and prevent their binding to RNA polymerase!
What is an example of an anti-sigma factor?
Anti-SF = FlgM Protein
–> Binds to sigma-28 (involved in timing of expression of genes involved in flagellar assembly)
Regulation of sigma factors is useful in global gene regulation for…
Controlling the TIMING of global gene expression
When anti-SFs are eliminated…
an ENTIRE regulon can quickly be turned on!
