Intro To Gene Regulation Flashcards
How is genetic expression regulated in prokaryotes?
- Regulate gene expression in response to environmental conditions
- E. coli has about 4,000 possible polypeptide chains encoded for on 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
Contrast constitute and inducible genes
- Bacteria have efficient mechanisms to turn transcription of genes off and on in response to environment and cellular activity
- nutrients available in the environment?
- specific nutrients require specific degradation enzymes
- nutrients available in the environment?
- Cellular activity including: replication, cell division, protein synthesis
- Are there enough nucleotides in the cell for replication?
- Are there enough amino acids in the cell for protein synthesis?
What are inducible genes?
Enzymes which are synthesized only when required based upon the chemical makeup of the environment are called inducible enzymes from inducible genes which are inducible expressed
- Turning off inducible genes referred 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 rare called Constitutive enzymes from constitutive genes which are constitutiveoy expressed
- Typically low level, constant expression
- Some gene products of constitutive genes regulate the expression of inducible genes
What is inducible gene regulation?
A gene (or group of genes) can have both positive and negative control as seen with the metabolism of lactose in E. Coli
What is negative control of inducible gene regulation?
Genetic expression occurs unless it is shut off by some sort of a regulator molecule
What is positive control of inducible genes?
Genetic expression occurs only if a regulator molecule directly stimulates RNA production
What are the 3 regulator molecule uses of prokaryotic cells?
Repressors
Activators
Inducers
What distinguishes repressors and activators from inhibitors?
Proteins produced in the cell which bind to DNA regions close to the genes that they will control- repressors and activators
Inducers-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?
these are DNA sequences on the same strand as the polycistronic cluster (cis-acting or cis-elements)
What are trans-acting elements?
These are the protein molecules that bind cis-acting sites
How can trans-acting elements regulate the transcription of structural genes?
Trans-acting elements can positively or negatively regulate the transcription of the structural genes
- Negatively by turning off transcription
- Positively by turning on transcription
What is a promoter?
- RNA polymerase recognize the promoter pribnow box and bind to it
- the RNA polymerase will scan along the DNA till it finds the transcription initiation(start) site for the structural gene
What is an operator?
- The operator is a sequence of DNA downstream of the promoter which is recognized by a repressors protein
- If the repressor protein in bound, RNA polymerase can not synthesize RNA
Explain the transcription of the structural gene
- RNA polymerase will bind to the promoter and transcribe the structural genes in a single unit (polycistronic RNA) including:
- Lac Z, Lac Y and Lac A
- the single RNA is translated into three genes products
- Entire gene cluster functions to provide rapid response to the presence or absence of lactose
Explain the catabolic conversion of the disaccharide lactose
- lactose can be metabolized for energy by bacteria
- First step is the cleavage of lactose to galactose and glucose
- The enzyme is B-galactosidase
- A minor activity of B-galactosidase is the conversion of lactose to allopactose
-Glucose can be metabolized directly but galactose must be converted glucose-6-phosphate before it is metabolized by the bacteria for energy
What is the preferred food source of bacteria?
Glucose
Who discovered lactose metabolism in bacteria?
Francois jacob and Jacques Monod
Explain how lactose metabolism in E. Coli is regulated by an inducible system
If glucose becomes depleted in the environment and lactose is present
-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 is converted to allolactose)
Remember Lac operons
What are operons?
- In bacteria, genes that encode enzymes with related functions tend to be organized in clusters
- these genes clusters are often under the coordinated control of a single regulatory region, entire region is called an operon
Explain the Lac operon
3 enzymes required to metabolize lactose
-B-galactose, permease and transacetylase
The 3 genes are called structural genes and they are clustered together in a tip to tail fashion(polycistronic)
- one regulatory region, promoter and operator
- one repressor gene, lacl which encodes for a repressor protein.
What are the 3 proteins of the Lac operon?
Lac Z, Lac Y and Lac A
What is the function of lacZ?
Encodes for B-galactosidase, an enzyme that converts the disaccharide lactose to monosaccharides glucose and galactose
What is the function of Lac Y in the operon?
Encodes for permease, an enzyme that facilitates the entry of lactose intolerance the bacterial cell
What is the function of Lac A in the Lac operon?
Encodes for transacetylase which may be involve in the removal of toxic byproducts of lactose digestion from the cell
What happens when there is no lactose?
Lac. Operon is repressed
Explain the Lac Operon repression
- Repressor protein binds to the operator
- RNA polymerase binds to promoter.
- RNA polymerase is inhibited by the repressor bound to the operator
- No transcription, no enzymes
Absence of lactose= repressor protein binds to operator, blocking transcription
Explain Lac Operon expression
- 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 when glucose is present in E. Coli?
Glucose is the preferred sugar in E. Coli, so when it is present, no need to express genes in the Lac operon
What is positive control of the Lac operon?
Genetic expression occurs only if a regulator molecule directly stimulates RNA production(that molecule is cyclic AMP)
Why is glucose the preferred sugar in E. Coli?
Lactose metabolism is not as efficient as pure glucose
What happens within bacteria when there is lactose but no glucose?
B-galactosidase will cleave galactose and glucose
The glucose portion is metabolized easily to glucose 6-p
The galactose must go through a long process to be converted to glucose 6-p
What are the effects of glucose and cAMP on the cell?
- If a cell runs out of glucose, a small molecule (cyclic AMP) is produced by activate adenyl cyclase
- cAMP is a ‘hunger signal’ that permits the expression of genes that break down other sugars, including lactose
- cAMP binds the activator protein CRP (cAMP receptor protein) or CAP (catabolic earth activator protein), which can then bind lacP to help activate transcription
What is the correlation between glucose and cAMP concentrations?
Inversely proportional
Explain 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.
- High transcription of the structural genes is stimulated
Explain Catabolite activation and repression
- 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
- In the presence of glucose, cAMP levels decrease, cAMP-CAP complexes are not formed, and transcription is not stimulated
Why is gene regulation more complex in eukaryotes than bacteria?
All eukaryotic cells contain the same genetic information. Muscle cells, neurons, etc., are different due to different expression of the genes
Gene regulation results in multiple different cell types
Contrast prokaryotic and eukaryotic gene expression
Prokaryotes:transcription and translation take place in cytosol and are coupled
-regulation at transcription and sometimes translation and modification
Eukaryotes: transcription in nucleus and translation in cytosol
- Regulation at many levels
- Chromatin compaction must be decreased for gene expression
- RNA must be processed for transport to cytosol
- Extensive protein modification
What 2 structural features of eukaryotes distinguish them from prokaryotes?
- Eukaryotic genes are situated on chromosomes that occupy at distinct location
- Eukaryotic DNA is combined with histones and non histone proteins to form nucleosomes that are wound into 30 no fibers which form more. Compact structures: chromatin
What are the functions of chromatin structures in eukaryotes?
Regulates gene expression:
Compact chromatin structure inhibits transcription, replication, and DNA repair
How can karyotypes be performed without Geimsa stain?
- Karyotypes can be performed with fluorescent probes
- instead of Geimsa staining, each chromosome has probes that will color each chromosome
What are functions of fluorescent probes?
- karyotypes
- the fluorescent probes also work on interphase DNA and you can see that chromosomes occupy particular regions in the nucleus
- best use of metaphase SKY FISH is looking for chromosome rearrangement particularly in cancer cells
What are chromosome territories?
Interphase DNA fills the nucleus
- Nucleus has territories for chromosomes
- Chromosome structure is continuously rearranged
What are the functions of chromosome territories?
- Transcriptionally active genes are moved to the edges of chromosome territories
- these regions contain high concentrations of active RNA polymerase and transcription regulatory proteins
- transcription factories on the edges of territories where a high concentration of transcription proteins are found
Where are Cis-acting sequences located and what do they do?
Cis-acting sequences are located in the same chromosome as the gene that they regulate :
- Promoters: core promoters, proximal promoters and distal promoters
- enhances
- silences
What do trans-acting elements do?
They bind to specific DNA regions (cis-acting DNA) located in and around genes and also found some distance away
- these sequences do not themselves regulate transcription
- they position regulatory proteins to these regions
- regulatory proteins (or small RNA molecules) can stimulate or repress expression
Trans-acting factors like DNA binding proteins can(or small RNA molecules) ……
Influence the expression of a gene on any chromosome
What is the core promoter gene?
This is immediately upstream of a gene
- there is no universal core promoter through many elements are covered
- INR is the transcription start site
- TATA box is about 30 NT, where the TATA box binding protein will bind
- BRE is recognized by transcription factors (TAFs)
What is the proximal promoter region
A eukaryotic CIS element that is upstream of a gene
DNA sequence-specific transcription factors will bind GC and CAAT box
What are Distant promoter regions of a eukaryotic gene?
These are enhancers can be found on either side of a gene, close by or up to 1 million base pairs up
-enhancer proteins that bind here can increase the rate of transcription
What are the CIS elements of a eukaryotic gene?
- core promoter gene
- proximal promoter gene
- distal promoter gene
What does transcription factor II D contain?
TFIID contains a protein that recognized the TATA box called TATA box binding proteins
What is the main function of the pre-initiation complex?
Formation of pre-initiation complex (PIC)
Explain Basal Transcription Machinery
- TFIID (and 13 other proteins called TAFs/TBP associated factors) are recruited
- Other general transcription factors (GTF) assemble on the promoter region as a platform for RNA polymerase II
- A mediator protein complex can help regulate transcription of the gene
- Fully formed PIC mediates unwinding of the promoter DNA at the start site of transcription
- RNA is elongated using DNA template
- This is Basal transcription and basal transcriptional machinery
What are Coactivators?
Some genes require coactivators which act as a bridge between activator proteins and the basal transcriptional machinery (not shown here)
What are the function of activator proteins?
Proteins bound to enhancer regions can interact with the mediator protein and TAFs to regulate gene transcription
What are the functions of enhancer regions?
Even when located far away from basal transcriptional machinery can come to close proximity by looping
What are silencer Regions?
Even when located far away from basal transcriptional machinery can come to close proximity by looping
What are repressor proteins?
Repressor proteins bound to silencer regions can interact with mediator protein and TAFs to regulate gene transcription
What are the function of co-repressors?
Some genes require co-repressors which act as a bridge between repressor proteins and the basal transcriptional machinery
What was the ENCODE DATA project?
- In 2003, the National human genome research institute launched the ENCODE project, the Encyclopedia of DNAelements project
- To identify all functional DNA sequences that lie within the 3.2 billion bases of human DNA
- Less than 2%DNA consists of protein-coding genes
What would be the reason of finding the non coding human DNA?
Finding the non coding DNA is not junk DNA as 80% is biochemically active
- 400,000 enhancers and silencers
- 100,000 promoters
- 7,000 small non coding RNA
- Enhancer RNA
- SNPs have been found in the above regions may predict susceptibility to disease
