Lecture 9 - regulation of gene expression Flashcards
At what points can bacteria control gen expression?
- control of transcription initiation
- mRNA stability
- control of translation
- post translational control (protein modification and degredation)
When is regulation most effective and efficient?
Earlier in the expression pathway
Transcriptional regulatory proteins
DNA binding proteins that increase/decrease expression of proteins by sensing changes and altering gene expression
Where are regulatory proteins encoded?
- either adjacent to the sequence they regulate…
- or far away if it regulates multiple genes (regulon)
Major regulatory strategies
- repression: transcription inhibited by repressor proteins that block RNA pol from binding promoter
- activation: transcription requires an activator protein to begin
How do repressor proteins usually work?
Cause steric inhibition that prevents RNA pol from accessing promoter
How do activator proteins usually work?
Binds adjacent to the promoter to stabilize the area for RNA pol binding
How are regulator strategies modulated?
Small molecules increase/decrease affinity of regulatory proteins for their operator sequences
Two examples of transcriptional repression mechanisms
- Repressor binds DNA and represses target gene; inducer causes repressor to release
- Repressor-corepressor complex binds DNA and represses target gene; corepressor removal causes repressor to release
LacZ
aka beta-galactosidase; breaks bond between galactose and glucose
LacY
aka lactose permease; allows import of lactose into cytoplasm
Steps of transcription induction of lac operon
- LacI repressor binds operator and prevents RNA pol from binding to lac promoter
- Lactose converted to allolactose by low levels of beta-galactosidase (LacZ)
- Allolactose binds LacI repressor and reduces its affinity for operator
Example of transcriptional activation mechanism
Activator binds ligand (inducer) –> complex binds regulatory sequence and activates target gene –> removing ligand stops transcription
Activation of lac operon by cAMP-CRP is an example of…
transcription induction via inducer ligand (cAMP)
Steps of lac operon activation via cAMP-CRP
- cAMP is made from ATP in low-energy conditions
- cAMP forms complex with cAMP receptor protein (CRP)
- cAMP-CRP complex binds promoter and interacts with RNA pol to increase transcription
- increased transcription of proteins for alternative metabolic pathways
Derepression of the trp operon
- when trp levels exceed cellular needs, excess tryptophan acts as co-repressor for TrpR
- trp operon transcription is repressed –> decreased synthesis of tryptophan
- when trp becomes limiting, is dissociates from TrpR –> TrpR falls off operator and trp transcription resumes
Why is repression of transcription important?
Cell wants to limit energy usage from unnecessary protein production
Is transcription repression complete?
No. Every so often, repressors will dissociate and produce small amounts of the transcript.
How are transcription and translation coupled?
Polysome structure: Multiple RNA pols make multiple transcripts that are being translated by multiple ribosomes per transcript
How do multiple ribosomes translate one mRNA strand?
Once the first ribosome moves down the mRNA and re-exposes the ribosome binding site and start codon, another small unit can bind.
What happens to transcription of the trp operon when trp is limiting?
- When trp is limiting, ribosomes pause at a trp-trp codon sequence in the leader peptide sequence because there aren’t as many tRNA molecules available with trp attached.
- Ribosome pausing causes mRNA strand to adopt a hairpin antiterminator structure; no interference with RNA pol activity
- Transcription is allowed to continue
What happens to transcription of the trp operon when trp is plentiful?
- When trp is plentiful, ribosomes move quickly through the trp-trp codon sequence in the leader peptide sequence because there are many available tRNA molecules with trp
- Since ribosome is not paused to obstruct the strand, crosslinking occurs with a neighboring portion of the mRNA molecule –> terminator conformation is made with a stem-loop structure right behind RNA pol
- Stem-loop structure signals transcription termination
Relationship between trp levels and trp operon transcription
- high trp –> low trp operon transcription
- low trp –> hig h trp operon transcription
Where is the trp-trp sequence located on the trp operon?
Between the leader region and the region encoding trp operon products
- if no transcription terminator, whole operon is transcribed
- if transcription terminator is made, operon is not transcribed
What E.coli operons mimic the ribosome stalling method of transcription regulation seen in the trp operon?
his, leu, and phe
Riboswitch
Region of mRNA that regulates transcription of the mRNA molecule through the binding of small molecules
Example of a two-component signaling system
Sensor kinase + phosphatase
How does the sensor kinase + phosphatase system regulate transcription?
- Environmental signal binds sensor kinase in the membrane
- Kinase phosphorylates response regulator
- Phosphorylated regulator binds operator and prevents RNA pol from proceeding
- When environmental signal is lower, phosphatase dephosphorylates regulators faster than kinase can phosphorylate –> regulators fall off and transcription can proceed
Examples of procedures regulated by two-component systems
- PhoQ/P: regulates Mg transport and virulence (Mg and phosphate usually travel together and tell the cell when it is inside a host cell)
- NarX/P: regulates nitrate respiration (alternative metabolic pathway)
How do alternative sigma factors work?
Recognize different -35 and -10 sequences to regulate expression of genes for different stress-management strategies or different lifestyles
How do numbers of different sigma factors differ between bacterial species?
More possible environmental changes = more kinds of sigma factors to account for the changes
- Mycoplasma genitalium (lives in our genital tract only) has one sigma factor because it lives in a constant environment
- Steptomyces coelicolor has over 60 sigma factors because it lives in soil
What sigma factor controls the heat shock response in E. coli?
Sigma 32 aka sigma H
What gene encodes sigma H?
rpoH
Is sigma H present in low temperatures?
- Sort of. The mRNA transcript of rpoH is present, but the secondary structure at low temperatures hides the ribosome binding site; very little sigma H can be made
- any sigma H that is made is degraded by DnaK or at least held away from the core RNA pol to prevent transcription of heat shock genes
Sigma H heat shock response at high temperatures
- At high temperatures, rpoH mRNA is denatured and ribosome binding site is exposed –> translation of sigma H is possible
- At high temps, DnaK prioritizes refolding of denatured proteins; sigma H is allowed to bind with core RNA pol and direct it to transcribe heat shock genes
Example of post-transcriptional regulation by small RNAs
- Positively acting small RNAs: either stimulate translation by unmasking ribosome binding site or by stabilizing mRNA (prevent RNase from degrading it)
- Negatively acting small RNAs: inhibit translation by occluding ribosome binding side or by stimulating degradation of mRNA (create RNase binding sites)
What is the stringent response to amino acid starvation?
Global rapid starvation response in bacteria. Responds to sudden decrease in AA’s by decreasing synthesis of ribosomes.
How does the stringent response to AA starvation work?
- AA deficiency results in ribosome pausing (no charged tRNAs available)
- RelA is activated and makes ppGpp from GTP
- ppGpp intteracts with RNA pol and decreases its affinity for rRNA promoters
- Result: ribosome production decreases and translation of proteins decreases; RNA pol transcribes more genes for AA synthesis
