Chapter 10 Lecture Outline Molecular Regulation

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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