Gene Regulation In Prokaryotes Flashcards
How is genetic expression regulated in prokaryotes ?
Bacteria regulate gene expression in response to environmental conditions
- E. Coli has about 4000 different possible polypeptide chains encoded for the genome
- Some proteins have only 5-10 molecules in the cell
- Others, such as ribosomal proteins and proteins in the glycolytic pathway can have as many as 100,000 copies in the cell
- Some proteins have very few copies (basal levels) which can be increased dramatically when required.
- There are regulatory mechanism to control the expression of genetic information
What are inducible genes?
Enzymes which are synthesized on,y when required based upon the chemical makeup of the environment are called inducible genes which are inducible expressed
- turning off inducible genes refferred to as repression or repressible - The presence of a specific molecule inhibits gene expression
What are constitutive genes?
Enzymes which are produced continuously, regardless of the chemical makeup of the environment are called constitutive enzymes from constitutive genes which are constitutively expressed.
- Typically low level, constant expression - Some gene products of constitutive genes regulate the expression of inducible genes
Explain inducible gene expression
Either positive or negative
A gene (or group of genes) can have both positive and negative control as seen with the metabolism of lactose in E. Coli.
Negative control- genetic expression occurs unless it is shut off by some sort of a regulator molecule
Positive control- genetic expression occurs only if a regulator molecule directly stimulates RNA production
What are the 3 types of molecules affecting expression in prokaryotic genes?
Prokaryotic cells use the following:
-Repressors
- Activators
- Inducers
Repressor sand activators are proteins produced in the cell which bind to DNA regions close to the genes that they will control
Inducers are small molecules either produced in the cell or taken up in to the cell, which can either activate or repress transcription, depending on needs of the cell at a particular time
What are regulatory regions?
Regulatory regions are DNA sequences on the same strand near the polycistronic cluster (promoter and operator)
What are activators and repressors?
The protein molecules that bind the regulatory regions upstream of the structural genes
- Can positively or negatively regulate the transcription of the structural genes
- Negatively by turning off transcription
- Positively by turning on transcription
How do structural genes play a role in prokaryotic DNA regulation?
- Structural genes encoding for enzymes are often organized in clusters (polycistronic) depending on function or pathway
- Transcription of these genes is under the control of a single regulatory DNA region located upstream of the cluster that they control.
What is a promoter?
- RNA polymerase recognizes the promoter sequence (pribnow box) and will bind to it
- RNA polymerase will scan along the DNA till it finds the transcription initiation (start) site for the structural gene
- RNA polymerase will begin transcribing the RNA of the structural gene
What is an operator?
- the operator is a sequence of DNA downstream of the promoter which is recognized by a repressor protein
- If the repressor protein in bound, RNA polymerase can not synthesize RNA
How is lactose metabolism in E. coli is regulated by an Inducible system?
- Francis Jacob and Jacques Monod were studying metabolism in E. coli
- Glucose is the preferred food source for bacteria
- Switched food source to lactose and studied what happened to gene expression
- 3 genes were expressed to protein, this is also called coordinate regulation
What is the preferred sugar source of bacteria?
Glucose
How is glucose metabolized in bacteria?
- Glucose is metabolized most efficiently by bacteria, first step is conversion to glucose 6-P
- If there is no glucose in the environment but there is lactose, an enzyme called B-galactosidase will cleave lactose to glucose and galactose
- The glucose portion is metabolized easily to glucose 6-P
- The galactose must go through many steps to be converted to glucose 6-P
Metabolism of lactose is not as efficient as pure glucose
What happens if glucose becomes depleted in the bacterial environment and lactose is present, what happens?
- The enzymes responsible for the metabolism of lactose increases rapidly from a few molecules to thousands per cell
- The enzymes responsible for lactose metabolism are inducible
- Lactose is the inducer (after it converted to allolactose)
What are Operons?
In bacteria, genes that encode enzymes with related functions tend to be organized in clusters
-These gene clusters are often under the coordinated control of a single regulatory region, entire region is called an Operon
How can lactose be metabolized for energy?
Lactose can be metabolized for energy by bacteria
-First step is the cleavage of lactose to galactose and glucose
- The enzyme is B-galactosidase (gene is LacZ)
- A minor activity of B-galactosidase is the conversion of lactose to allolactose
-Glucose can be metabolized directly but galactose must be converted glucose 6-phosphate before it is metabolized by the bacteria for energy
Describe the transcription of Lac operon structural genes
-RNA polymerase will bind to the promoter and transcribe the structural genes in a single unit (polycistronic RNA) including : lacZ, lacA, and lacY
The single RNA is translated into three gene products
Entire gene cluster functions to provide rapid response to the presence of absence of lactose
Describe the basics of Lac Operon
There are 3 enzymes required to metabolize lactose
- B-galactose, permease and transacetylase
These 3 genes are called structural genes and they are clustered together in a tip to tail fashion (polycistronic)
-lacZ, LacY and lacA
- One regulatory region, promoter and operator
- One repressor gene, lacl which encodes for a repressor protein
What is the function of lacZ?
Part of the Lac operon- encodes for B-galactosidase, an enzyme that converts the disaccharide lactose to monosaccharide glucose and galactosidase
What is the function of lacY?
Encodes for permease, an enzyme that facilitates the entry of lactose into the bacterial cell
What is the function of lacA?
Part of the Lac operon- encodes for transacetylase which may be involve in the removal of toxic by-products of lactose digestion from the cell.
How does presence of lactose affect the Lac operon?
No lactose around so you don’t need the structural genes synthesized
1. Repressor protein binds to the operator
- RNA polymerase binds to promoter
- RNA polymerase is inhibited by the repressor bound to the operator
Absence of lactose: repressor protein binds to the operator, blocking transcription
How does the presence of lactose effect Lac operon?
Lactose is present so you need the structural genes synthesized
Induction of the Lac Operon when lactose is present
1. Lactose is converted to allolactose
- Operator binding region of repressor protein is altered when bound with allolactose
- No binding occurs; transcription proceeds
- Structural genes transcribed
What happens if Lac operon has structural gene mutations?
lacA-, lacY- and lacZ-: structural gene mutations lead to non-functional proteins
What happens if lacP- gets mutated?
Non-functional promoter, RNA pol cannot bind so genes will not be expressed
What happens when lacO^C is mutated?
Non-functional operator, repressor cannot bind. Since the system cannot be shut off, this is a constitutive mutation
What happens if lacI- is mutated?
Non-functional represssor : unable to bind the operator to shut off transcription system always on
What happens if lacLs is mutated?
Super-repressor; unable to dissociate from operator. System always off
Mutation prevents binding to allolactose, so will not dissociate from operator
How does glucose effect Lac operon?
Therefore, when glucose is present, there is no need to express the genes in the Lac operon
Positive control
-genetic expression occurs only if a regulator molecule directly stimulates RNA production (that molecule is cyclic AMP)
What happens to bacteria if there is no glucose in the environment?
- If a cell runs out of glucose, a small molecule (cyclic AMP) is produced by active Adenylyl cyclase(converts ATP to cAMP)
- cAMP is a ‘hunger signal’ that permits the expression of genes that break down other sugars, includ8ng lactose
- cAMP binds the activator protein CRP (cAMP receptor protein ) or CAP (catabolite activator protein), which can then bind lacP to help activate transcription
Describe cAMP activation
In the absence of glucose, cAMP levels increase, resulting in the formation of cAMP-CAP complex
- cAMP/CAP complex binds to the CAP site of the promoter
- The binding of cAMP to the CAP protein inducing a conformational change to the complex such that it binds upstream of the promoter region of the Lac operon
- The DNA is stabilized allowing for RNA polymerase to bind with high efficiency
- High transcription of the structural genes is stimulated
RNA polymerase binds with high efficiency once activated CAP protein is bound
Summarize positive regulation
a) in the absence of glucose, cAMP levels increase resulting in the formation of a cAMP-CAP complex, which binds to the CAP site of the promoter, stimulating transcription
b) in the presence of glucose, cAMP levels decrease, cAMP-CAP complexes are not formed, and transcription is not stimulated
What are the kinds of regulation of Trp operon?
Transcriptional regulation
Translational regulation
What is the Trp operon responsible for?
The Trp operon is responsible for biosynthesis of structural enzymes required to synthesize the essential amino acid tryptophan
- if tryptophan levels are low, turn on expression to produce more
- if tryptophan levels are high, turn off the system
Explain the functioning of the Trp repressor
The Trp operon is another example of negative regulation of gene expression. Within the operon’s regulatory sequence:
- the operator is blocked by the repressor protein in the presence of tryptophan (preventing transcription)
- repressor protein fails to bind the operator in tryptophan absence(allowing transcription)
- The repressor protein is encoded by the TrpR gene, locat3d far from the Trp operon
What are the 5 structural genes of the Trp Operon?
Structural genes: Trp E, Trp D, Trp C, Trp B and Trp A
Trp P and Trp O are promoter and operator sequences
What does the leader sequence of the Trp sequence?
Leader sequence contains ribosomal binding site with multiple Trp codons and region called the attenuator which folds in one of two alternative RNA structures
What happens to Trp operon if Trp is absent?
- in the absence of tryptophan, an inactive repressor is made
- The inactive repressor can not bind to the repressor
- RNA polymerase will bind to the promoter and transcription of the structural genes will occur
What does the presence of Trp in Trp operon?
- In the presence of tryptophan, it binds to the repressor protein
- This causes an allosteric change to occur
- The complex now binds to the operator leading to repression of the operon
Explain the translational control of Trp gene expression
- Another method of control is through attenuation
- The first protein-coding gene in the Trp operon is the ‘leader peptide’ trpL, which contains adjacent codons for tryptophan
- Therefore, if tryptophan - tRNA is abundant in the cell, translation of this peptide will be quick
Explain the attenuation of the trp operon
- mRNA secondary structure is essential in this regulation
- If tryptophan is abundant, the ribosome will speed quickly through the Trp codons BUT
- the mRNA BEHIND the codons will form a stem-loop terminator structure, and RNA pol will fall off before it can transcribe trpEDCBA
Describe the most stable secondary structure for trp leader mRNA
The most stable secondary structure for Trp leader mRNA is shown in the given image
- Attenuation depends on the ability of regions 1 and 2 to form a hairpin loop with 3 and 4 forming a hairpin looping …
- 2 and 3 CAN form a hairpin loop but above is the preferred lowest energy conformation
Following the 3-4 loop is a string of U nucleotide
This is similar to the termination signal for RNA polymerase.
What happens when tryptophan is abundant?
RNA polymerase is making mRNA and the ribosome is making a peptide until…
- The ribosome pauses at the stop codon
- mRNA forms a 3-4 hairpin loop region followed by a run of UUUU
-RNA polymerase dissociates from DNA &
No more RNA is made
-Ribosome can’t make the 5 proteins without proper mRNA
What happens when tryptophan is rare?
RNA polymerase is making mRNA and the ribosome is making a peptide until…
- Until the ribosome stalls at a Trp codon because tryptophan in low in the cell
- While the ribosome wars for a loaded tRNA, the 2 to 3 stemloop structure is formed and no stop signal is made for RNA polymerase
- RNA polymerase makes a proper polycistronic mRNA for the Trp operon (mRNA for all structural genes) because there is no longer a 3-4 structure
Summarize Trp attenuation
Translational regulation
- If tryptophan is rare in the cell, the ribosome will pause at the Trp codons
- This pausing will prevent the formation of the terminator signal, and RNA pol will continue transcribing the trpEDCBA genes
