CH15- Genetics
Heterochromatin
-condensed DNA -transcription is repressed -underacetylated histones -methylated DNA -some parts of genome are shut off/ inactive and some are open
Euchromation
-uncondensed DNA -capable of transcription DNA can be expressed !!!!
Prokaryotic gene expression
1)Controlled by regulation of transcription. 2)Regulation involves repressor and activator proteins that block or help RNA polymerase bind to the promoter (transcription factors) 3) Small molecules regulate the activity of the repressor and activator proteins (cAMP, glucose, etc) Mechanism involving encoded proteins with related functions organized in clusters under coordinated control.
CAP binding sites
3 binding sites on CAP molecule: 1 binds cAMP, 1 a DNA sequence upstream of the lac promoter, and 1 RNA polymerase. CAP binding site is next to the promoter. Binding of CAP improves the binding of RNA polymerase to the promoter.
Regulation use by partial diploids
Adding I+ to I- restores inducibility by producing the normal wildtype repressor from the I+ gene. I is a trans-acting receptor and does not have to be next to the sequence it regulates. Adding O+ to Oc doesn't effect constitutive enzyme production because regulation depends on presence of O+ directly adjacent to the structural genes. O is a cis-acting receptor, must be next to sequence it regulates. Adding I+ to Is does not relieve repression of gene activity.
Negatively or positively
Binding of trans-acting molecule to the cis-acting site can regulate gene clusters either _______, or_______.
CCAAT
Binds to promoter Increases promoters ability to initiate transcription
CAP function
CAP activates expression of the lac operon. Exerts positive control when binding to the CAP site. Inhibits expression when glucose is present. "Transcription Factor"
Constitutive mutations
Cells bearing this type of mutation produces enzymes regardless of the presence or absence of lactose. These enzymes are produced continuously, inducibility is eliminated, and gene regulation is lost. "Transcription factors" Example: LacI- , LacOc
Promoters
Cis regulators that bind to proteins that regulate transcription initiation. Sequences adjacent to transcription start site and contains the "core promoter" aka polymerase binding site. Needed for basal level of transcription
Operons
Clusters with associated regulatory control are called ______, and transcription within these clusters is either inducible or repressed by the metabolic substrate or end products of the pathway
+ and -
Combinations of both___ and ____ regulatory mechanisms determines transcription of the lac operon.
I-
Constitutive mutation of I gene (repressor gene). It is close too Z,Y,A genes on the chromosome. Allows for continuous transcription because the repressor protein is altered or absent and it cannot bind the operator region. Transcription proceeds and the enzymes are produced in the absence or presence of lactose.
Oc
Constitutive mutation of the operator. Located adjacent to structural genes in the operator region of the operon. Allows for continuous transcription because the DNA sequence of the operator molecule is altered and the repressor molecule will not normally bind. Transcription proceeds and the enzymes are produced in the absence or presence of lactose.
Why are certain genes active in some cell types but not in others?
Each gene requires a specific set of transcription factors Different cell types contain different transcription factors. Genes use a mixture of transcription factors, some are active in some places and some are not.
Glucose
Ecoli favors _______, which inhibits transcription of lac operon. Ecoli can get more energy from this molecule than it could from lactose.
I gene
Encodes for a repressor protein that binds to the operator and blocks transcription. I gene is always active and repressor protein is always made in small amounts. Repressor protein has 2 binding sites, a portion recognizes operator sequence, and a portion of protein finds lactose. The repressor does not bind to the operator sequence in the presence of lactose, and transcription proceeds.
Repressible expression
End products of biosynthesis pathways, or presence of a specific molecule that inhibits gene expression and transcription is called _____.
Enhancer loops
Enhancers form DNA loops at a distance from the promoter to interact with general transcription factors. Influences stability and stimulates the rate of transcription initiation
Increase
Enzymes for lactose metabolism rapidly ______ in the presence of lactose. Thus, categorizing lactose enzymes inducible. Lactose is a tiny, regulatory circuit.
........
Eukaryotes have ability to switch from heterochromatin to euchromatin... and DNA can exist in these 2 states.
Other levels of gene control in eukaryotes.
Eukaryotes regulate gene expression in other than only transcription level. -They also have splicing/capping (regulated to make different proteins from different mRNAs) -Have more DNA and modify structural organization. -Regulation of transport - Most RNAs have greater half life than prokaryotics RNAS -RNA translation regulation--> proteins can be degraded
Gene regulation of eukaryotes
For development and tissue/ cell differentiation. Regulate their growth and division to occur at appropriate places in the body and during appropriate times of development. Regulation by internal factors like hormones (turning genes on/off), not by changing signals from the environment.
Negative control
Gene expression that occurs unless it is shut off by some regulator molecule
Catabolite repression with glucose.
Glucose inhibits activity of adenyl cyclase, which catalyzes the conversion of ATP--> cAMP. Decreased cAMP and CAP-cAMP complexes are not formed, CAP nor RNA polymerase binds efficiently, and transcription stops.
Metabolite repressor
Glucose is called a __________ because it represses metabolism of lactose.
Proximal Promoter
Has other sequences to get RNA to move to core promoter.
I-O+Z+/ F'I+
I+ is a trans-acting receptor and still produces the repressor molecule when added to I- bacterial chromosome. B-galactosidase is produced in the presence, but not absence of lactose.
Amount of B-galactosidase made:
If glucose and lactose are present, a 10x increase in B-galactosidase is made. If glucose is present without lactose, hardly any B-galactosidase is made. If lactose is present without glucose, 1000x B-galactosidase is made.
cAMP
Important intracellular signaling molecule in many cells. Formed by adenyl cyclase when ATP levels are low. Internal phosphodiester bond is made and used to regulate different substances. CAP binds with camp, and ecoli can now grow on many diff substances that may be outside the cell. Each operon has its own specific repressor.
nothing
In eukaryotic genes, what would happen if a single base substitution occurred between the stop codon and the polyA signal?
+1
In prokaryotes, promoter recognizes and binds to ____.
Catabolite repression without glucose.
In the absence of glucose, catabolite-activating protein (CAP) binds to cAMP. The CAP-cAMP complex binds to the CAP binding site of the promoter region, facilitating binding of RNA polymerase in the promoter region. Transcription occurs
Steroid Hormone
Induced gene expression All cells are exposed to hormone ex: estrogen, testosterone, corticosteriods, retinoic acid --> Each turns on transcription of specific genes in specific organs. Cells that respond to hormones have receptors for the hormone--> sends signal to nucleus
SV40 Control Gene
Infects monkeys, control gene comes on and is expressed in high levels Enhancers contain multiple sequence elements--> different transcription factors bind and work to produce high levels of gene expression
lac- mutants
Isolate mutants with nonfunctional enzymes. Mutants that fail to produce active B-galactosidase (lacZ-) or permease (lacY-) are unable to use lactose as an E source and called _____.
Steroid Hormone Receptors
Lipid soluble hormones, the receptor is a transcription factor.
Regulation of Transcription
Most important level of control. 3 RNA polymerases control transcription: Pol I- rRNA Pol II- mRNAs-- proteins Pol III- 5s rRNA and tRNA Multiple RNA polymerases used that transcribe different genes, each polymerase recognizes different promoters to know which genes are there and which aren't. Poly2 transcribes the biggest set of genes and has the biggest promoter. Eukaryotes have alot of transcription factors to get high level of gene expression.
Is
Mutant repressor molecule that cannot interact with the inducer, lactose. Results in the repressor always binding to the operator sequence. Transcription of these structural genes are permanently repressed. Operon is uninducable--> cannot make b-galactosidase in presence of lactose
Operon Model
Negative control--> transcription occurs when the repressor molecule fails to bind to the operator region. In the presence of lactose, the sugar binds to the repressor molecule which induces a conformational change, reducing the repressor molecule from acting with the operator region of the DNA. Transcription of genes are indirectly stimulated. Without lactose present, the repressor molecule normally binds to the DNA sequence of the operator region, which inhibits the action of RNA polymerase and repressing transcription.
I+OcZ+/F'O+
O+ is a cis-acting receptor and adding it to Oc does not change the regulation of the enzyme. Transcription proceeds because the repressor never binds to the operator. B-galactosidase is produced in the presence or absence of lactose.
Coordinated control
Polycistronic mRNA results in this type of regulation of all 3 genes, since a single messenger RNA is translated to all 3 gene products simultaneously.
trp operon
Produces enzymes (5) needed for tryptophan synthesis. Tryptophan biosynthesis is an anabolic pathway (buliding up) that is regulated by the end product itself (tryptophan)
Inducible
Prokaryotic enzymes that are only expressed when a specific chemical or substrate is present
Constitutive
Prokaryotic enzymes that are produced continuously, regardless of the chemical makeup of the environment.
Transcription Factors
Proteins that bind specific DNA sequences and facilitate or inhibit the binding of RNA polymerase. Ex: Lac and trp repressors and the CAP protein. Operator sequence is recognized by the repressor protein. (inverted repeats)
Overlap
RNA polymerase and Repressor binding sites _________. When repressor is bound, RNA polymerase is blocked.
Positive
Regulation by CAP is under _______ control.
cis-acting site
Regulatory control sites found on DNA and upstream from the operon it operates on. These _____, or regulatory regions, bind molecules (trans-acting molecules) that control transcription of the gene cluster.
Metallothionein gene
Regulatory sequences in the promotor. Gene expressed specifically in the liver and its expression is up-regulated by heavy metals and corticosteriod hormones. Activity is turned on when heavy metals are present or by stress hormones. Liver specific transcription factors: AP 1/2/4 General TF and liver specific TF are activated a little at a time.
Negative
Repressible system is under __________ control.
Maximal transcription of structural genes.
Repressor bound by lactose (so it doesn't repress lac operon transcription) AND CAP must be bound to CAP-binding site.
Enhancers
Sequences needed for high level of transcription, can boost level of gene expression. Cis regulators can be located on either side of gene, distant or within the gene. They are responsible for time and tissue specific gene expression. Not all genes have enhancers; only needed if tissue specific or gene needs to be expressed at high levels (ex: thymidine kinase)
TATA box function
TATA binding factor recognizes and binds to the sequence, TATA associating factor is bound to complex protein to facilitate RNA polymerase binding. Transcription factor upstream needed to bind first. Enhancer sequences help stabalize the RNA Pol II at the TATA box.
GC
Transcription fact found in proximal promoter region
Repressors
Transcription factors that decrease level of transcription initiation.
Activators
Transcription factors that increase level of transcription initiation.
Positive control
Transcription only occurs if a regulator molecule directly stimulates RNA production
corepressor
Tryptophan is known as a ________. When tryptophan is present, resultant complex of repressor-tryptophan binds to operator, reducing transcription.
Cooperative binding
When both cAMP-CAP and RNA polymerase are bound to lac promoter region, a tightly bound complex is formed, called ___________.
I-O+Z-/ F'I+
Z- is a mutation in the Z gene resulting in no enzyme production or in the formation of a nonfunctional enzyme. No B-galactosidase is produced in the presence or absence of lactose.
Structural, polycistronic
______ genes of the lac operon are lacZ, lacY and lacA. Mapping studies showed that these genes are closely linked (Z-Y-A). All these genes are transcribed as a single unit, resulting in a _______ mRNA.
Genetic expression
_______ is regulated by mechanisms that control transcription, mRNA stability, translation and posttranslational modification.
Specific Transcription Factors
bind to specific promoter and enhancer sequences to promote binding of RNA polymerase to core promoter at the TATA box. Transcription factors bind to each other to build the complex on the promoter. Binding of transcription factors regulate gene expression.
trp repressors
blocks transcription in the presence of tryptophan, enzymes needed for tryptophan synthesis are not produced. "Transcription Factor"
lactose metabolism
catabolic (breaking down)pathway that is regulated by the availability of substrate (lactose).
Silencers
cis-acting transcription regulatory element with short DNA sequences that represses the level of transcription initiation.
permease
lac Y encodes AA sequence _____, an enzyme that facilitates movement of lactose into bacterial cells
B-galactosidase
lac Z gene encodes for _________, an enzyme who's role is to convert lactose to galactose and glucose. This conversion is essential for lactose as a source of energy in glycolysis
