1. Chapter 10 Lecture Outline
Molecular Regulation

2. Microbial Gene Organization
DNA-dep. RNA polymerase binds
Helps align mRNA in ribosomes
Promotor
Leader
Activator/Operator
Structural genes
(transcribed)
On-Off switch
Operon
Multiple genes transcribed from one promoter
Genes are transcribed together
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

3. Regulating Gene Expression
Microbes monitor two different compartments
Intracellular
Concentrations of vitamins, sugars, amino acids, nucleotides
Control of de novo synthesis and degrading enzymes
Extracellular
What type of environment (e.g., pond water, gastrointestinal tract, in a host cell)
Adjustment of gene expression for best protection/invasion
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

4. Regulating Gene Expression
Microbes must sense their environment
Receptors on cell surface
Receptors must transmit information to chromosome
Signals from activated receptor alter gene expression
Change transcription rate
Change translation rate
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

5. Response to Conditions Outside of the Cell
Sensing the Environment
Two-component signal transduction
Sensor kinase protein in plasma membrane
Binds to signal
Nutrient
Chemical cue
Activates itself via phosphorylation
Cytoplasmic response regulator
Takes phosphate from sensor
Binds chromosome
Alters transcription rate of multiple genes Or
Activator
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

6. Microbial Control of Gene Expression Occurs at Multiple Levels
Alteration of DNA sequences
E.g., phase variation
Control of transcription
Operators = sequences on DNA / repressors = proteins binding to DNA
Activators = sequences on DNA / activators = proteins binding to DNA
Control of mRNA stability
Translational control
Post-translational control
Modifying protein activity by chemical modification like phosphorylation, methylation, acetylation etc.
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

7. Control of Gene Transcription
Regulatory proteins bind to DNA regulatory sequences that control gene transcription
Operators: binding of regulator (repressor) down regulates expression of target genes
Activators: binding of regulator (activator) increases expression of target genes
Regulatory proteins (regulators) bind to small molecular weight compounds (ligands)
Different regulators bind to different ligands
Once ligand is bound to regulator the ability of regulator to bind to DNA is altered
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

8. Gene Regulation via Repressors
Repressors (protein) bind to operators (DNA sequence)
Upon binding to the operators repressors inhibit gene transcription
Two scenarios are possible:
Active repressor is removed from operator by ligand (inducer) binding
Gene was off  now gene on
Gene induction
Inactive repressor binds to operator after binding of ligand (co-repressor)
Gene was on  now gene off
Gene repression
If ligand diminishes and not available for repressor binding, repressor releases from operator
Gene derepression
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

9. Gene Regulation via Repressors
Lactose operon
Tryptophan operon
Active
Inactive
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

10. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
The E. coli lac Operon
Lactose (milk sugar) is used as nutrient
Cannot pass through plasma membrane
Lactose permease allows entry
Proton motive force used to move lactose inside cell
Must be converted to glucose to be digested
b-galactosidase converts lactose to glucose
Humans also make b-galactosidase
If not, person is lactose-intolerant
Inducer
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

11. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
The E. coli lac Operon
The lacZ gene encodes b-galactosidase
The lacY gene encodes lactose permease
Need both proteins to digest lactose
Role unclear
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

12. The E. coli lac Operon Repressor protein LacI blocks transcription
Active repressor binds to operator
Repressor responds to presence of lactose
Binds inducer (allolactose) or DNA, not both
Add lactose in the absence of b-galactosidase  allolactose accumulates  repressor falls off operator
allolactose
© 2008 W.W. Norton & Company, Inc. MICROBIOLOGY 1/e
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

13. Click box to launch animation
The E. coli lac Operon
Animation: The lac Operon
Click box to launch animation
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

14. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
Concept Quiz
What is the phenotype of an E. coli strain with a mutation in the lac operon operator such that Lac repressor could never bind to the operator?
The strain never transcribes lac genes.
The strain transcribes lacZ and lacY only when lactose is present.
The strain always transcribes lacZ and lacY, even without lactose present.
Answer: C
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

15. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
Trp Operon
Cell must make the amino acid tryptophan
Requires many proteins, made from one operon
When tryptophan is plentiful, cell stops synthesis
Trp repressor must bind tryptophan to bind DNA
Opposite of lac repressor
Repressor + Tryptophan
Transcription repressed
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

16. Gene Regulation via Activators
Activators proteins typically bind poorly to activator DNA unless an inducer is present
If inducer concentration diminishes activator protein can no longer bind to activator DNA and gene transcription ceases
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

17. Sigma Factor Regulation
Allow coordinated expression of large gene sets
Normally degraded
Under stress no degradation
Binding to core RNA Polymerase
Sigma factor/RNA polymerase complex binds to various heat-shock genes
By controlling s factor expression large gene sets can be controlled
s factors regulate transcription of all genes
s70 initiates transcription at most genes
Alternative s factors used for special cases
Sporulation in B. subtilis—Bs s28
Stationary phase—s38
Heat-shock and stress response—s32
Flagellar synthesis—s28
Control of ratio of s factors determines global control of protein synthesis
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

18. Sigma Factor Regulation
Temperature-sensitive mRNA structure
Allows translation of s32 only at high temperature.
Proteolysis rapidly removes s factors
Rapid turnover allows more exact control
s70 degraded rapidly at 42°C
Synthesis of proteins that inhibit s factors
Anti-s factors block s activity until needed
Anti-anti-s factors respond to environment
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

19. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
Small Regulatory RNAs
Many intergenic regions encode small untranslated RNA (sRNA)
Small RNAs regulate transcription or stability of specific mRNA nmolecules
Does not require protein translation
Economical regulation
Antisense RNA base-pairs to mRNA
Usually prevents translation
Until removed via endonuclease
Universal method of gene control—found in all creatures
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

20. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
Quorum Sensing
Cells work together at high cell density
V. fischeri becomes bioluminescent
Many bacteria form biofilms
Exoenzyme production
Toxin production
Send signal chemical to other cells
Chemical accumulation = high cell density
Autoinducer
Binds to sensor in cell
Sensor activates transcription
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

21. Microbial Communication through Quorum Sensing
Autoinducer diffuses into the medium where it accumulates. At threshold concentration AI diffuses back into the cell and binds to activator protein LuxR.
LuxI synthesizes the autoinducer homoserine lactone
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

22. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.
Peptides
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.