Chapter 18: Regulation of Gene Expression
Gene Expression
process by which information encoded in DNA directs the synthesis of proteins or RNAs that are not translated into proteins and instead function as RNAs
the trp operon
is a repressible operon
multicellular organisms: cells have specific jobs that
require specific proteins
rough ER->golgi->secretory vesicles->cell exterior
secretory pathway of protein synthesis and secretion
what are control elements?
segments of noncoding DNA that serve as binding sites for transcription factors, which help regulate transcription
Kozak sequence
a DNA consensus sequence called the _______ ________ has been shown to be optimal for translation initiation
gene cluster
a ______ _______ is a set of genes all with a similar function that are located in relatively close proximity to one another on the chromosome.
remodeling complex
a _________ ________ is a protein complex that disrupts the interactions between histone proteins and DNA
coactivators
an enhanceosome is a complex of proteins containing __________ or corepressers that act in concert to regulate transcription in eukaryotes
a lac operon is
an inducible operon
What are oncogenes?
cancer-causing genes that arise from a genetic change, leading to increase of proto-oncogene's protein product or in the activity of these molecules.
multiple inputs regulate
transcription
"GU" at the 5' donor splice site "AG" at the 3' acceptor splice site
"GU-AG" rule
when glucose levels increase
-cAMP levels fall -CRP detaches from the lac operon -transcription of the lac operon is reduced
genes "on"
-constitutively expressed
1-2 b- galactosidase
In E. coli, without the lactose how many B- galactosidase is produced
What get cAMP to AMP?
Phosphodiesterase
What level does most translational regulation occur at? What is this mechanism sensitive to?
Most known examples of translational regulation operate at the level of translational initiation - mechanism is highly sensitive to the structure of the target mRNA
Why are SAM and magnesium important riboswitch ligands?
SAM- important because it is involved in methylation - Important to be able to sense the presence of SAM to know about the methylation and presence of methionine in the cell DAM methylation: occurs after DNA synthesis Magnesium: important to transport - Don't make a metal à need to transport it
Soluble NSF Attachment protein REceptors
SNARE
acetylation
The facts that TAFs contain HATs and bromodomains allows for cascades of __________ reactions to occur
repressor
The operator sequence regulates transcription by, depending on the proteins that it is bound to, determining whether RNAP can continue moving down the antisense strand to transcribe structural genes. The proteins that bind the operator and prevent transcription are referred to as ___________ proteins
1956; Jacob, Monod and Lwoff
The operon was discovered in year ____________ by ______________,_______________,________________
If the corepressor is present, repressible protein is also, what will happen to the operon?
The operon will be turned off because the repressor protein is on that turns the activator on
This is the anti-termination hairpin, because it prevents the formation of termination hairpin making the RNA polymerase to continue transcribing the entire operon
What do you call the 2-3 Hair pin? Why?
Transcription Termination Sequence, because once it forms (3-4) , RNA polymerase will disassociate/ fall off from the DNA and transcription of the structural genes of the operon will not occur
What do you call the 3-4 structure? why is it called that?
This means that their products are constantly produced at a fixed rate
What do you mean by : "Genes are not regulated but are instead *constitutive*"
How can face of the helix dependence be identified?
identified by construction of small insertion and deletion mutants
alternative splicing
_______ _______ of a single RNA transcript gives rise to different proteins; increases diversity of proteins
cAMP
________ stimulates transcription by binding to CAP, which then binds the promoter sequence and increases transcription.
post-translational control
_________ __________ of gene expression refers to the control of protein activity, stability, localization and interacting partner molecules
silencers enhancers
_________ and ___________ are regulatory gene sequences
mRNA
_________ in the nucleus must be processed before leaving for the cytoplasm
regulatory proteins
__________ ______ specific to cell type control intron-exon choices by binding to regulatory sequences within the primary transcript
footprinting
__________ is a technique used to identify specific binding site of a repressor, polymerase, or other protein on the DNA
activators promoter
a mediator is a protein that facilitates the interaction between the __________ bound to enhancers and the RNAP bound to the ________.
in eukaryotic transcription regulation, activators
activate transcription
during development, signals b/w cells
activate transcription factors that begin the differentiation process
tropomyosin
an example of normal alternative splicing is alternative splicing of the _____ gene
enhancer promoter
an insulator is a region of DNA that defines the range of interaction between the _______ and the _______
gene cluster
an operon is a _______ _______ in which all of the genes are controlled by the same promoter, such as the proteins for the metabolism of lactose in the lac operon.
post translational modifications (PMT)
attaching biochemical functional groups
constantly transcribed
attenuation is a control mechanism used by genes that are:
post-transcriptional gene control
attenuation is an example of:
DNA methylation can condense
chromatin
heterochromatin euchromatin
chromatin that is highly condensed and in which the DNA is not available to be transcribed is referred to as _________ whereas chromatin that it less highly condensed is referred to as _________
(multicell organisms) Each cell type has the same DNA content, but uses
different sets of genes to generate cell specific proteins
difference between cell types result from
differential gene expression
Leucine zipper
dimers result from leucine residues at every other turn of the a-helix. When the a-helical regions form a leucine zipper, the regions beyond the zipper form a Y-shaped region that grips the DNA in a scissorslike configuration
How do different sets of activators come to be present in two cells? just name the two ways:
distribution of cytoplasmic determinants, and different inductive signals.
heart development
during _____ ________ miRNAs has essential roles such as the regulation of tissue specific transcription factors important for cardiogenesis
combinatorial gene regulation
during development, different cell types are created due to the different combination of TFs (gene regulatory proteins).
heterochromatin
highly condensed regions, transcriptionally inactive
increased
if sequence four was removed from the trp attenuator then transcription would be ________ (increased/decreased)
decreased
if sequence three of the trp attenuator was altered so that it could pair with sequence four but not sequence two then transcription would be ________ (increased/decreased)
aminoacyl-tRNA
in the first step of protein synthesis, _____________ binds to vacant A site
intron
in the fourth step of RNA splicing, the _______ is released
spent tRNA
in the fourth step of protein synthesis, the ______ _______ is ejected and the ribosome is reset
cAMP Receptor Protein (CRP) and Catabolite Activator Protein (CAP)
this is a positive regulator and relies on the Presence of cAMP which depends on the metabolic activities of the cell
a repressible operon is usually
on
the trp operon is ____ when tryptophan is _______
on, absent
DNA double helix
once RNA pol and TFs assemble at the promoter, _____ _______ ________ is pulled apart so that transcription can begin
co-translational transport
ribosomes with mRNA attached are targeted to the ER, where translation occurs
consensus sequences
signal where splicing is to occur
Repressors bind to?
silencers, slow down rxns
imprinting
the phenomenon in which genes are silenced through methylation depending on which parent they are inherited from is called ________
Repressor
this is a regulatory proteins that acts as off switches for transcription by preventing the binding of RNA polymerase
Tryptophan operon
this is a negative repressible transcriptional control (because of the absence of tryptophan)
Repression
this is a response to the overabundance of an end product
Genes
this is effectively turned on all the time and codes for enzymes that the cell needs in fairly large amounts for its major life processes
Transcription terminator
this is located near the end of the leader sequence and before the tryptophan E and is composed of hairpin loop in the RNA
Activator
this is regulatory proteins that acts as an on switch for transcription
B- galatosidase
this is responsible for the lactose hydrolysis
Leader peptide
this is the part of the leader transcript that codes for a short polypeptide of 14 amino acids
Promoter
this is the site in DNA which the RNA polymerase recognizes as an initiation signal to indicate where transcription of RNA begins
Operator
this is the site in DNA which the repressor would bind
Attenuator
this is the site where the transcription usually stop
Positive Control
this is when the regulatory protein CRP stimulates transcription
Negative control
this is when the regulatory protein, lactose repressor STOPS the transcription (Ex. When the lactose is present the lac repressor is falls off, but when the lactose repressor is bound, RNA polymerase cannot bind and genes cannot be transcribed)
Inducible
this means that the operon is turned on by the substrate of the enzyme for which the structural genes code
inducible enzymes
synthesis is induced by chemical signal (ex. allolactose); an enzyme whose transcription can be stimulated by an abundance of its substrate (as opposed to repressible enzyme); usually in catabolism.
target membrane SNAREs
t-SNARES
repressor
the CAP-cAMP complex stimulates transcription in the absence of the lac _________ protein.
For the Lac Operon what repressor does it need?
the Lac Repressor
C-chain
the ________ of insulin is removed by cleavage
TATA box
the __________ _____ is a common promoter element in eukaryotes
chromodomain
the __________ is the region of the remodeling complex, which disrupts histone DNA interactions, that binds the methylated chromatin
DNA methylation
the addition of a methyl group to DNA
DNA methylation
the addition of methyl groups (—CH3) to bases of DNA after DNA synthesis; may serve as a long-term control of gene expression.
If a particular operon encodes enzymes for making an essential amino acid and is regulated like the trp operon, then
the amino acid acts as a corepressor.
in eukaryotic transcriptional regulation (EukTR), RNA polymerase requires
the assistance of transcription factors to initiate transcription
histone acetylation
the attachment of acetyl groups to certain amino acids of histone proteins; usually makes the gene more accessible for transcription
leader
the attenuator sequence is located within the _______ sequence of the trp operon
Prokaryotes
this organisms organize collection of genes into operons
Repressor Proteins
this protein responds to cell conditions to actively repress the RNA polymerase beginning transcription
Gene Expression
this refers to the combined processes of transcription and translation of genetic information to make a functional protein
Feedback inhibition
this stop the cells from performing a needed chemical reaction
Lactose Permease
this transports lactose across the cell wall
Repression
this turns off the operon when the nutrient is no longer required
Negative Regulation
this type of regulation involves a repressor protein which responds to cell condition to actively repress transcription of the genes by binding onto the DNA at the operator site
Chomosomes
threadlike structure within nucleus that contains genetic info that is passed on from generations
Helix-turn-helix (homeodomain)
three different planes of the helix are established and bind to the grooves of the DNA
regulation of gene expression allows cells
to efficiently control cellular resources
gene expression/regulation allows cells
to maintain homeostasis
genes are turned on and off in response
to signals from their external and internal environments
Where are TF's translocated?
to the chromatin
DNA helix
top strand rungs 5 to 3. bottom runs 3 to 5. - main thing here is to know that adenine and thymine form 2 hydrogen bonds while cytosine and guanine form 3
1. recruit RNA pol II to the promoter 2. position and aid RNA polymerase binding to the promoter
transcription factors are required to:
Downstream promoter element (DPE)
transcription initiation factors and their consensus sequences
inducible operon
usually off, but can be stimulated (induced) when a specific small molecule interacts with a regulatory protein (example lac operon)
vesicle membrane SNAREs
v-SNAREs
Corepressor
The ligand that controls the activity of the tryptophan acts not as an inducer but as a _________________
Repressor proteins
The negative transcriptional control system codes for?
What would happen if the repressor was only bound to a single operator?
There may still be some interference with transcription to some extent but the promoter wouldn't be bent
Coactivators
These "adapter" molecules integrate signals from activators and perhaps repressors
What is the lac and gal operons examples of? What is the normal type of regulation for these kind of operons?
They are catabolic operons which are usually managed under negative inducible systems
3000 b-galactosidase for 2-4 mins
In E.coli with lactose how many B-galactosiase is formed
two
In a hairpin loop The leader RNA has how many tryptophan codon?
What happens when RNA polymerase reaches the pause site? What does the ribosomes progress through the trp codons determine?
A. RNA pol. pauses at 1:2 pause site and allows the ribosome to load onto the leader RNA B. The ribosomes progress along the RNA eventually bumps RNA polymerase off the pause site The ribosome's progress through the trp codons determines whether the terminator helix or anti-terminator helix forms
What regulation happens in the absence of tryptophan?
Absense of tryptophan: The trpR gene is transcribed as an aporepressor and transcription of the operon is able to occur
histone code
According to the ________ _________, specific post transcriptional modifications to histone proteins leads to particular gene expression outcomes
Activators bind to?
Enhancers, speed up rxns
What enzymes breaks the Glycosidic Linkage?
B-Galactosidase
loop structure
when miRNA is initially transcribed, we see it undergo some processing, but because it has RNA sequence and reverse compliment sequence, it forms back on itself, forming a:
Maternal effect genes=
when mutant in mother, mutant phenotype in offspring even if offspring genotype is normal. mRNA protein products of maternal effect genes are put in egg when it's still in the ovary.
attenuation
when the gene product is required, regulatory proteins bind to the RNA chain and interfere with _______, allowing transcription of a complete RNA molecule
genes are turned "off"
when they are not transcribed
genes are turned "on"
when they are transcribed and the mRNA is translated into protein
cAMP-CRP complex
when this is bound, it helps push RNA polymerase to clamp down the mRNA form into a closed form RNA Polymerase and start transcribing z,y, and a genes
RUNX2
mutations in ______ causes supernumerary teeth
what are the functional RNAs
-rRNA -tRNA -miRNA
What is a group of distal control elements called?
Enhancers
What is a cluster of genes(often together) under the control of a single promoter?
Operon
A site
aminoacyl-tRNA enter the:
bacteria use operators to
control transcription
exon splicing
removal of introns from pre-mRNA
myoD is a
"master regulatory gene" -that encodes a transcription factor that commits the cell to becoming a skeletal muscle
Lac Operon: Glu and Lac control
(+Glu/+Lac): Operon off; no repressor as lactose will be an inducer to the repressor, while glucose will prevent CAP protein from binding due to catabolite repression. No CAP means that the operon is not active. (+Glu/-Lac): Lac repressor bound as Lactose is absent; Operon is off bc now repressor is bound and CAP protein still cannot bind due to catabolite repression. (-Glu/-Lac): Repressor is bound as lactose is absent; CAP protein is able to bind to cAMP (Glucose is not present), which allows for the complex to bind to the activator site. Operon STILL OFF as the repressor prevents activation. (-Glu/+Lac): Repressor is not bound as lactose present and will act as an inducer. With glucose not present, the cAMP can bind to CAP protein, then binding to activator site; OPERON IS ON as there is not repressor preventing this
COP II
(red) vesicular trafficking protein ER --> cis-golgi (anterograde)
Gene Activation (Steroid Hormone)
(Nonpeptide) hormones enter cell via diffusion -Binds hormone receptor in the cytoplasm -Binding causes the receptor to translocate to the nucleus -In nucleus, the receptor acts as a transcription factor and binds to promoter elements of genes that are regulated by that hormone (these promoter elements are called HORMONE RESPONSE ELEMENT)
COP I
(blue) vesicular trafficking protein golgi --> cis-golgi --> ER (retrograde)
Clathrin-coated vesicles
(green)Vesicles form at the plasma membrane to the TGN; essential for endocytosis
DNA binding site for protein
*Affinity of a particular transcription factor for target sequence is a million fold higher than other DNA sequences. *DNA target site usually palindrome repeat (inverse) to which 2 subunits of regulator sequence bind cooperatively. *Not possible yet to know which DNA sequence a given transcription factor will bind. *Involves Helix-turn-Helix, Zinc Fingers, HMG Box Motif *DOUBLING the contact site, squares the association constant; 2^amount of doubled sites= Association constant*
RNA polymerase II Binding to a Promoter
*Basal (general) transcription factors: Needed at every RNA polymerase II promoter *DNA binding transactivators: Bind to enhancers and facilitate transcription *Co-activators: Act indirectly by bridging between DNA binding transactivators and complex compose of RNA polymerase II and the general transcription factors.
Operon
*Cluster of genes involved in the same biochemical pathway under control by one promoter sequence. -Genes are transcribed together as a polycistronic mRNA. *Operon includes the promoter region which contains next to a binding site for RNA polymerase, a binding site for activator, and one for the repressor. -Repressor binding sequence: Operator -When repressor binds to operator= preventing transcription
Steroid hormone receptor protein
*DNA binding domain: Alpha helical region inserted in the major groove- Will bind to DNA of interest *Transactivation domain: Related to transcription activation *Dimerization domain: OPTIONAL (make dimer with another protein) *Hormone binding site
SWI/SNF Complex
*Enzymes that use ATP to remodel nucleosomes on the DNA* -Actively move or displace nucleosomes -Create hypersensitive sites in DAN and stimulate binding of transcription factors -SWIne SNifF= SWI/SNF
Gene Silencing
*Example: X-chromosome inactivation -Occurs in females and imprinting -Inactivation of a certain gene dependent on whether it is inherited from the father or the mother.
Ferritin and Transferrin IRE Regions
*Ferritin mRNA has the IRE in the 5'-untranslated region. -Make hairpin structure in 5'UTR *Transferrin receptor mRNA has the IRE located at the 3'-untranslated region. -Makes hairpin structure in the 3'UTR *In the IRE-binding protein the site for iron binding and for RNA binding overlap. *Excess iron causes dissociation of binding protein from the IRE (won't bind). *Binding of the IRE-binding protein to the 5'-IRE blocks translation of ferritin mRNA *Binding of the IRE-binding protein to the 3'-IRE stabilize the transferrin receptor mRNA.
HOXD13 Mutation
*HOXD13 gene encodes for Homeobox protein Hox-D13. -When mutation arrises in the gene (homeobox), the homeodomain will have Protein-DNA binding issues. *Can lead to synpolydactyly*
HATS and HDAC
*Histone acetyl transferases and Histone deacetylases- Enzymes that covalently modify the core histones of nucleosomes* -Acetylation status of amino-terminal domains of histone H3 and H4 determines that affinity between histones and DNA. HAT:Co-activator HDAC: Co-Repressor
DNA-Protein Interaction (Bonding type)
*Hydrogen bonding *Hydrophobic interactions -Methyl groups of Thymidine
Side Effect of Tamoxifen
*INCREASED risk for endmeyrial cancer because in endometrial cell the co-activator is still available to bind to tamoxifen/estrogen receptor complex. -Though tamoxifen is a co-repressor in estrogen producing sites, it is the opposite in endometrial cell production.
DNA Methylation
*In Prokaryotes: A in GATC is methylated *In Eukaryotes: Cytosine bases in CpG are methylated. *Cytosine in 5'-CpG-3' is substrate for DNA methylases *Methylated DNA is associated with gene-silencing -Demethylation leads to DNA activation
Dnase hypersensitive sites
*In chromosomal DNA, DNase marks region that are devoid of histone proteins, but have DNA-only regions *The regions with no histones allow for binding of transcription factors. *Region is upstream of the transcription initiation site *Hypersensitive sites vary from selected genes- cell type specific function *DNase is a in vitro lab tool that cuts DNA 3'-5' (endonuclease)*
Aconitase
*Iron Sensor* High iron conditions: Will not bind Low iron condition: It will bind to ferritin to inhibit it, while enhancing the production of transferrin
Levels of Iron
*Iron Starvation: Ferratin is NOT made, but transferrin receptor is made. IRE binds to neither -Low iron concentrations the iron responsive element binding protein will stabilize the transferrin receptor protein mRNA leading to more translation of it. *Iron Excess: Ferratin is made, but transferrin receptor is NOT made.IRE binds to both! -Excess presence of iron causes iron-responsive element binding protein to dissociate from the iron responsive element so that ferritin mRNA translation is not inhibited.
Protein-Protein Interaction Domains
*Leucine Zipper and Helix-Loop-Helix*
Leucine Zipper
*Leucine-rich regions in two proteins interact to form a dimer -Coiled coil of Alpha helices -Adjacent to zipper region is a basic amino acid segment that interacts with DNA= bZIP domain. -Zippers can be homeodimers (same) or heterodimers
Helix-turn-Helix
*Many bacterial regulator proteins interact with DNA via helix-turn-helix motifs in the protein. *Recognition helix: The helical region interacting with the DNA.
Zinc Fingers (Function)
*Multiple zinc fingers substantially enhance binding by interacting simultaneously with the DNA. *GATA sequence present in DNA promoter site: GATA family of zinc finger transcription factors recognize this sequence. *Zinc does NOT directly interact with DNA *Interaction between a SINGLE Zinc finger and DNA is weak (multiple make better binding) *Involved in genes for embryogenesis, ertyropoiesis, muscled development, hemoglobin synthesis, etc.
Translation Feedback: In Ribosomal protein operons
*Normal growth conditions: Allows for ribosomal protein operon to make proper mRNA and translate to ribosomal proteins; rRNA gene will translate to rRNA, bind to ribosomal proteins and make ribosomes *Adverse growth condition: When rRNA gene is signaled to stop transcription, less rRNA is formed. While toe ribosomal protein operon transcribes and translates ribosomal proteins, they will accumulate and not have the rRNA needed to form ribosomes. As a result, the excessive ribosomal proteins will bind to its mRNA (still transcribing from ribosomal protein operon) to prevent further translation of the mRNA. *Interaction of the proteins occurs with hairpins in the 5'UTR or the 3'UTR of the mRNA.*
Ways to Measure Gene Expression
*Northern Blot: RNA put on agarose gell and blotted; probe can help identify a specific sequence -Nuclear rRNA will be at different lengths due to RNA processing; shortest strand will be mature mRNA within the nucleus *RT-PCR: Reverse transcriptase PCR: Retrovirus can be verified via reverse transcriptase, then PCR process, and gel electrophoresis. *DNA microarray: Spots in test will represent specific mRNA that are being expressed or unexpressed.
Leader region of trp mRNA
*Nucleotide sequence of the 5'-end of trp mRNA includes a short Open Reading Frame (ORF) that encodes a 14 amino acid peptide containing two Trp residue and untranslated attenuator region: 3 regions that can form hairpin formations. *Downstream of ORF: Hairpin formation favored is 2:3 (Proximal hairpin); attenuation occurs at 3:4 hairpin formation (Distal hairpin)*
Maintenance of gene repression
*Occurs with linked DNA and histone methylation* -MeCP Proteins (methylated Cp-G binding Proteins 1 and 2) binds to methylated CpG dinucleotides. -MeCP2 recruits HDAC activity to remove acetyl groups from histone and induce chromatin remodeling. -MeCP2 then aids in the methylation of H3K9 (Lysine 9 in histone H3) -HP1 (heterochromatin protein 1) binds to methylated H3K9 and to itself= condensing of the chromatin.
Low Tryptophan Levels
*Promote hairpin 2:3 formation* -Enables uninterrupted transcription -Ribosome pauses at the Trp codon in sequence 1 -Formation of hairpin between sequence 2 and 3 PREVENTS attenuation *Sequence 2 is no longer available to form attenuator structure with sequence 4. -Transcription will continue *Proximal hairpin is favored: Complete transcription of operon*
High Tryptophan Levels
*Promote hairpin 3:4 formation* -Enables Rho-factor independent termination of transcription -When the levels are high, ribosome quickly translates sequence 1 -Sequence 2 is blocked from translation before sequence 3 is transcribed. -As transcription continues, attenuation occur by hairpin structure of 3 and 4. *Distal hairpin is favored: premature termination of the transcript*
Hormone Response Element
*Short sequence of DNA within the promoter of a gene that is able to bind to a specific hormone receptor complex= Regulate Transcription* -50 nuclear hormone receptor proteins known and mediate the effect of steroid, thyroid, and retinoid acid proteins. -Usually nuclear hormone receptor protein is a dimer -HRE= Palindromic (inverted) repeat -The bound nuclear hormone receptor will then recruit a co-activator or co-repressor to the promoter region, resulting in chromatin remodeling.
Classes of regulator Proteins
*Specificity Factors *Repressors *Activators
Co-Activators in General Transcription
*TFIID: Besides general transcription factor activity, it also works as a coactivator.(Transactivators on Enhancer-> TFIID (the co-activator)-> TBP) *Mediator Coactivator: Required for both basal and regulated transcription at promotors used by RNA polymerase II -Binds to C-terminus (CTD) of the large subunit of RNA polymerase II an stimulates phosphorylation of that region by TFIIH (Transactivators on Enhancer->Mediator-> CTD of RNA Polymerase II) *Coactivators involved in chromatin remodeling
Zinc Fingers (Structure)
*Zinc (2+) binds with 4 cystines (C) or 2 cystines+ 2 histidines *2 amino acids in Alpha-Helical region, and 2 amino acids in Beta-Sheet region. *About 20 amino acids long
What is the regulation of gene expression? What is the regulation considered if it occurs at the first stage of when an RNA is made?
- the process by which the expression of genes is turned on and off at different times and under different conditions - If it occurs at the first stage of when an RNA is made it is considered to be transcriptionally regulated.
What is the E. coli lac operon important for? What are the structural genes and regulatory elements present and what are their functions?
- E. coli lac operon encodes enzymes used for lactose utilization - encodes the following structural genes required for lactose utilization: lacZ: encodes B-galactosidase which cleaves lactose into glucose and galactose lacY: permease for lactose brings lactose inside the cell lacA: transacetylase (function unclear) - Lac I is the repressor protein - lacO is the operator sequence - lacp is the promoter
What is an inducer and a co-repressor?
- Inducer: signal that increases gene expression by either inactivating a repressor or by activating an activator - Co-repressor: signal that decreases gene expression by activating a repressor or inactivation of an activator
What is the difference between positive and negative transcriptional regulation at initiation?
- Positive: initiation is controlled by an activator that is required for efficient initiation by RNA polymerase at the promoter. - Negative: expression is controlled by a repressor that binds to a repressor binding site or operator sequence in the DNA that prevents the initiation of transcription by RNA polymerase
What is a repressor protein? How does it generally work and how can its activity be mediated?
- Repressor: a protein that mediates negative regulation - binds to a repressor-binding site or operator sequence in the DNA that prevents the initiation of transcription by RNA polymerase - Activity can be mediated by the binding of a small molecule: inducer or co-repressor
enhancers cam be located
- distantly from the promoter -upstream, downstream, within an intron
low glucose, high lactose
-cAMP level high -active CRP -abundant lac mRNA synthesized -RNA polymerase binds and transcribes
Amino Acids involved in DNA binding
-All have long side chains) *Arginine (R) *Lysine (K) *Asparagine (N) *Glutamine (Q) *Glutamate (E) "RKN EQ!"
Hairpin loop
-GC-rich sequence in prokaryotes that RNA polymerase recognizes to terminate transcription
Coregulators
-Intracellular enzymes (eg. JNK/MAPK) -Extracellular signaling factors
Types of intracellular enzymes
-Mitogen-activated protein kinase & cJun N terminal kinase
What are the most variable regulations of DNA expression:
-developmental signals for embryonic growth and differentiation -environmental cues for adaptation (responses to signaling factors -cell cycle control, pathogenesis), etc
high glucose, high lactose
-inactive CRP -cAMP level low -little lac mRNA synthesized -RNA polymerase less likely to bind
DNA methylation can reduce (pro and euk)
-reduce methyl cytosine in euk -reduce methyl adenine and cytosine in prok
an operon is the entire stretch of DA that includes
-the operator -the promoter -and the genes they control
What do activator and repressor competition control?
-tissue specificity -hormone activation -temporal window of expression
post-translational transport
-translation occurs in the ribosomes in the cytosol -once protein is made, it is transported to its functional cellular location
2 steps of post transcriptional regulation
1) miRNA binds to a target mRNA 2) if bases complementary, mRNA is degraded if match is less complete, transcription is blocked
How do transcription factors alter the transcription rate? (General)
1) position RNA polymerase 2) separate DNA strands 3) release RNA polymerase from promoter *this slows or speeds up the process*
Repressor specific transcription factors (STF) work in two ways:
1. turning off transcription even when activators are bound, by binding between the general transcription factors (GTF's) and the enhancer. 2. Not allowing binding of activators, by blocking that site.
Where are the 2 operators on the galactose operon?
1. upstream of the promoter 2. within the first gene (galE)
Explain how enhancers and activators interact with transcription factors to affect gene expression:
1. Activator proteins bind to distal control elements grouped as an enhancer in the DNA, upstream of the gene. 2. A DNA bending protein brings the bound activators closer to the promoter. GTF, mediator proteins, and RNA pol are nearby. 3. The activators bind to certain mediator proteins and GTF's, helping them form an active transcription initiation complex on the promoter.
1. anticodon 2. amino acid attachment site
2 key sites on tRNA:
One of the noncoding RNAs that regulate gene expression is microRNA. Explain two modes of action of microRNAs (miRNAs):
1. Enzyme cuts each hairpin from the primary RNA transcript. 2. Second enzyme, the Dicer, trims the loop at the single stranded ends of the hairpin. 3. One strand of the double stranded RNA is degraded. The other strand then forms a complex with one or more proteins. 4. The miRnA can bind to any target mRNA that contains at least 6 bases of complementary sequence. 5. If miRna & mRNA bases are complementary all along their lenth, mRnA is degraded. If match is less complete, translation is blocked.
movement of the DNA in the genome is a problem and will cause a proto-oncogene to become an oncogene because...
1. It could have a new promoter that caused excess production. 2. It could be transcribed over and over to make multiple copies.
Control Mechanism
Ensures that the genes are active only when their products are required
B. subtilis trp operon: What are the 2 levels of control?
1. Transcription attenuation: kill the message 2. Translational control: make sure the message isn't read
1. enzymatic modification of the histone N-terminal tails 2. ATP-driven chromatin remodeling complexes
2 mechanisms to change chromatin structure:
Steps of the cell stimulating pathway:
1. growth factor binds to receptor in plasma membrane. 2. Signal is related to a G protein called Ras that needs GTP bound to it to relay signal. Hyperactive Ras protein (product of oncogene) issues signals on its own. 3. The last kinase that the signal is relayed to activates a transcription activator that turns on one or more genes for proteins that stimulate the cell cycle.
6 Simple steps of colorectal cancer development:
1. loss of tumor suppressor gene in normal colon epitherial cells 2. small benign growth (polyp) occurs 3. Activation of ras oncogene 3. loss of DCC tumor suppressor gene 4. Larger benign growth occurs (adenoma) 5. loss of TSG p53 5. more mutations 6. malignant tumor forms (carcineroma)
the signal hypothesis
1. signal sequence is synthesized 2. signal binds to SRP 3. SRP binds to receptor 4. Growing Protein enters ER 5. Signal sequence is removed
How much of human genes actually code for protein?
1.5%
constitutive regulated
2 types of secretion:
nucleosome
1st stage of DNA compaction
Enzyme classes that play a role in chromatin remodeling
2 classes *HATs and HDACs *SWI/SNF Complexes
What percentage of the genes of a typical human cell are expressed at any give time?
20%
Transcription Factor Types
2500 types, this is 10% of the total 25,000 proteins
Regulation of gene expression at translational level (Types)
3 Ways to regulate: *Protein Kinases *Translation feedback *Secondary mRNA structures (Iron-responsive elements-IRE)
stop
3 codons are ___ codons and do not represent amino acids, but instead cause termination
-acetylation -methylation -phosphorylation
3 enzymatic modifications of the histone N-terminal tails:
1. activation 2. conjugation 3. ligation
3 steps of ubiquitination
general transcription factors
6 types, eg. TFIIA and TBP
20
61 of the 64 possible codons code for _____ amino acids
the trp operon is _____ when tryptophan is ______
off, present
cis Golgi network (CGN)
A collection of fused vesicular tubular clusters arriving at the Golgi from the ER
What has to bind to the repressor protein in order for the repressor to bind to the operator turining the Operon off?
A corepressor
cytoplasmic determinant
A maternal substance, such as a protein or RNA, that when placed into an egg influences the course of early development by regulating the expression of genes that affect the developmental fate of cells.
signal recognition particle (SRP)
A protein-RNA complex that recognizes a signal sequence as it emerges from a ribosome and helps direct the ribosome to the endoplasmic reticulum (ER) by binding to a receptor protein on the ER.
transcription factors
A regulatory protein that binds to DNA and affects transcription of specific genes.
signal sequence
A short sequence of amino aids, usually found at the N-terminus of a protein being translated, that directs the ribosome and its associated mRNA to the membranes of the rough ER where translation will be completed.
What occurs in histone acetylation? How does it affect gene expression?
Acetylation of histone tails promotes loose chromatin structure (beads on a string), permitting transcription. Acetyl groups are attached to lysines in histone tails, so their positive charges are neutralized and histone tails no longer bind to neighboring molecules.
HAT TAF TFIID
Acetylation of histones within nucleosomes increases the transcription of the DNA bound because the associated between the histone protein and the DNA becomes less favorable. Acetylation reactions are carried out by histone acetyl transferases (_______s). One of the TATA binding protein Associated Factors (_____s) that makes up the general transcription factor ___________ is a HAT.
negative.
Acetylation, phosphorylation, and ubiquitination all increases the _________ charge of the histone proteins and cause them to repel the DNA. As a result, the DNA and the chromatin is less bound to itself and access to the DNA by transcription machinery is increased.
Signal Molecules
Acting on a repressor *Co-repressor: Enhances the binding of the repressor *Inducer: Reduces the binding of the repressor to the operator. Acting on an Inducer *Co-Activator: Enhances the binding of the activator. *Repressor: Reduces the binding of the activator to the promotor.
What is an activator and how does it work? How can activity be mediated?
Activator is a protein that mediates positive regulation Often increases the tightness of binding of RNA polymerase or manipulates the DNA strands to help RNA polymerase bind Activity can be mediated by binding of a small molecule -> inducer
Gene-specific regulatory transcription factors
Activators and Repressors
What happens to the ability of regulation when 5 base pairs are added or deleted between the 2 operator sites ( araI2 and araO2)? 10 base pairs?
Adding or deleting 5 base pairs: this adds half a turn of the helix which positions the second AraC further linearly and on the opposite side of the DNA helix from the other AraC --> blocks repression Adding or deleting 10 base pairs: - Adds a full helix turn which extends the linear distance but the first AraC can interact with the second one
What gets ATP to cAMP?
Adenyl Cyclase
Enzymes
All metabolic reactions are catalyzed by?
What is the corepressor for the lac repressor protein?
Allolasctose
Specificity Factors
Alter specificity of RNA polymerase for promotor -Sigma factor in bacteria -TBP protein in eukaryotes
How can folding impact transcription attenuation? Where does this occur and what structures are made?
Alternative folding of the leader RNA into either a terminator helix (folding between B and C) or an anti-terminator helix (folding between A and B)
What happens to the rpoH transcript when the cell is subjected to heat shock ( an abrupt increase in temperature)?
An abrupt increase in temperature melts the secondary structure of the rpoH mRNA and allows translation --> results in rapid increase in the amount of sigmaH The RNA polymerase with sigmaH recognizes promoters of genes that help the cell respond to heat shock
protein
An operon is a group of genes that are located next to each-other in a chromosome, have similar functions, and are regulated by the same promoter. The reason that operons occur is to cut down on the amount of unnecessary ________ that is produced
What makes the repressor protein?
Another gene on the DNA genome
leader
Attenuation is a mechanism of transcription regulation in prokaryotes. It is deponent on the rate at which a short peptide is synthesized is synthesized from a ______ sequence located between the structural genes and the operator.
translation
Attenuation would not be a reasonable mechanism of gene regulation in eukaryotes becase transcription and __________ do not occur at the same time
allolactase
Bacteria preferentially metabolize glucose for energy. however, when lactose is present it triggers transcription of the lac operon which encodes the enzymes necessary for lactose metabolism. The physiological inducer of transcription of the lac operon is ____________
Why would E. Coli have lots of cAMP around?
Because it ran out of glucose to make ATP, so turns to the cAMP, which can activate the Catabolite Activator Protein, which calls the RNA Polymerase to transcribe and make enzymes that could get more glucose by maybe break glycosidic bonds between the galactose and glucose that make up Lactose.
Why is Allolactose called in the Lac operon?
Becuase it binds to active repressor in the Lac activator and makes it inactive
What happens when LacI repressor is bound to operators 1 and 3 (or 1 and 2)?
Bent DNA Stabilizes the LacI and helps to prevent the RNA polymerase from binding to the promoter
How does the TF regulate the specific cell transcription?
Binding to regulatory sequences: -Enhancer -Silencer *these are far away from the gene*
How does CAP binding to the CAP binding site of the E. coli ara operon alter the DNA structure formed? What does this possibly do?
By binding to the CAP binding site, CAP may help to open the DNA loop created when AraC binds to araO2 and araI2 --> may help prevent AraC binding to araO2 and araI2 and facilitate AraC binding to araI to activate transcription from pBAD
How does CAP protein tune up transcription?
By recruiting RNA Polymerase
What is only active when bound to cAMP?
CAP
When is CAP activated as a DNA binding protein? When does this happen? What does CAP do?
CAP is active as a DNA binding protein when the the secondary messenger cAMP (cyclic AMP) is high cAMP is high only when glucose is low CAP enhances RNA polymerase binding in the absence of glucose
What tunes up transcription?
CAP protein (Catabolite Activator Protein) by recruiting RNA Polymerase
What binds at the CAP site?
Catabolite Activator Protein
What does CAP stand for?
Catabolite activator protein
How can functionally related genes with different promoters and at different places in the chromosome be affected all at the same time?
Change in chromatin structure to make a whole group available or unavailable
Transcriptionally Inactive Chromatin
Chromatin Structure: Highly condensed (heterochromatin) DNA Methylation: Methylated, including at promoter region Histone Acetylation: Deacetylated histones
Transcriptionally Active Chromatin
Chromatin Structure: Open, extended conformation= Euchromatin DNA Methylation: Unmethylated, especially at promoter region Histone Acetylation: Acetylated histones
What 5 things can be regulated in Eukaryotic Cells?
Chromatin, Transcription, Post-Transcription, Translation, Post-Translation
Cis and Trans
Cis: Regulatory elements (promotors and enhancers) are on the same chromosome as the transcribe gene Trans: Activator or repressor proteins reach the point of action by diffusion -Enhancers may be thousands of base pairs away from transcription initiation site and make contact with the initiator site through transactivators. -DNA looping allows for interaction between distant DNA sites and HMG (high mobility group) proteins that are involved in DNA-dependent processes (Transcription, etc) *HMG proteins causes the DNA to bend!*
What would occur if the repressor of an inducible operon were mutated so it could not bind to the operator?
Continuous transcription of the operon's genes
What aid promoters in Transcription?
Control Elements
Chromatin
Clusters of DNA, RNA, and proteins in the nucleus of a cell
Coactivators: Chromatin remodeling complex
Coactivators have HAT activity or bind to aceytlated Lys -HATs catalyze transfer of acetyl group from Acetyl Coenzyme A to various Lys and Arg residues in amino terminal regions of the histone core proteins (weakens histone-DNA bonds). -Effects of acetylation of histone tails: Prevent chromatin from folding in compact structures, thus maintains the euchromatic state (loosening DNA-histone interaction) -Acetylation of histone tails means that increased access of specific DNA regions to chromatin remodeling complexes proteins. -SWI/SNF can come in; promoter region will ultimatey become available
AP-1 transcription factor complex
Complex: c-Jun + c-Fos targets a range of genes involved in cell cycle progression, cell survival and apoptosis
Lactose Operon
Consists of structural genes involved in lactose hydrolysis -Repressor is inactivated by binding to allolactose (the INDUCER) -Lactose operon can only work when it is properly activated by CRP (cAMP responsive protein) ; aka CAP protein. -CRP binds to the activator site when cAMP is bound (the co-activator). -Glucose lowers intracellular cAMP levels by inhibiting adenylyl cyclase, causing CAP to lose its bound cAMP- catabolic repression due to glucose effect. *ALWAYS some expression of lac operon*
What is the purpose of the E. coli gal operon and what are the structural genes and their functions?
Conversion of galactose into glucose GalK - kinase to phosphorylate galactose GalT - transferase that transfers galactose 1 phosphate to UDP glucose displacing the glucose to make UDP-galactose GalE - epimerase that concerts UDP-galactose to UDP-glucose
What do the structural genes do in the ara operon?
Convert L-arabinose to D-xyulose-5-phosphate
3'->5'
DAN associates with mRNA 5'methyl cap and then degrades the mRNA in the _________ direction
How do activators reach the promoters?
DNA LOOPING & BENDING enable activators to reach the promoter and bind with either DNA or mediator proteins
p53
DNA binding protein that uses protruding peptide loops
gene regulatory proteins transcription factors
DNA binding proteins are also called:
chromatin chromosomes
DNA exists as condensed structures called __________ or __________
octomeric
DNA is wrapped around an _______ histone protein core
heterochromatin
DNA is wrapped around histone Octamers calle nucleosomes. The nucleosomes are condensed into a protein/DNA structure called chromatin. Highly condensed chromatin is referred to as _______ and is not expressed very much because it is not available to the transcription machinery.
binding sites
DNA methylation acts as _______ _______ for other proteins which "read" the modification and recruit other and/or are associated with other proteins which can modify histones
DNA methyltransferases
DNA methylation is catalyzed by enzymes called:
CpG dinucleotide
DNA methylation occurs on cytosine nucleotides that are found next to a guanine nucleotide in the DNA sequence that are linked to form a ______ ___________ and forms a 5-methyl-cytosine
Types of TF
Diverse in nature, but several common structures are found. -Helix-turn-helix -Zinc fingers -Leucine zipper
Homeobox
DNA sequence found in genes that codes for the Homeodomain (the protein domain that will bind to DNA eventually).
What happens when point mutations are introduced to the riboswitch where the ligand binds?
Depending on the mutation you can either form the terminator or the antiterminator permanently but either way there is a loss in the ability to regulate expression
How can alternative RNA splicing result in different proteins derived from the same initial RNA transcript?
Different mRNA are produced from the same primary transcript, depending on what is treated as exons and what is treated as introns.
euchromatin
Dna is wrapped around histone Octamers to form nucleosomes that are then condensed to form chromatin. Chromatin that is relatively uncondensed allows its DNA to be accessed by the transcription machinery and is referred to as a __________
RNA polymerase
During attenuation, the growing RNA chain adopts a structure that causes it to interact with the _______ __________ in such a way as to abort its transcription
if glucose is scarce and lactose is present, then E.coli
E. Coli will turn on the lac operon to digest lactose
Glucose/lactose affect on E. coli
E. coli prefers Glucose as carbon source -Can utilize lactose in absence of glucose -When both are present: glucose is used preferentially, but utilization of lactose can still occur (beta- galactosidase expression).
B. subtilis trp operon: Each TRAP subunit binds a repeated ________ only if the subunit has ___________
Each TRAP subunit binds a repeated 3 base sequence only if the subunit has tryptophan bound
Explain how the LacI repressor protein uses different interactions in the repression of the E. coli lac operon.
Each symmetrical half of an operator binds a LacI monomer Simultaneous binding of the LacI tetramer to 2 operator sites allows each of the 4 subunits of the tetramer to interact with an operator site
Activator
Enhance RNA Polymerase-promotor interaction. -Regulation by an activator is called positive regulation. -Ligand can remove the activator protein, or can activate it. -Binds to ENHANCER
Epinephrine Gene activation
Epinephrine activates G-protein coupled receptors= Intracellular signaling (CANNOT enter cell and uses hormone receptors) -Activated G-protein binds to and activates adenylyl cyclase giving increasing cAMP level -cAMP binds to regulatory subunit of protein kinase A (PKA) and the released PKA catalytic subunits move to the nucleus. -PKA catalytic subunits phosphorylate the cAMP-Response Element Binding Protein (CREB) in the nucleus -P-CREB binds to cAMP response elements in the promotors of target genes -Then binds coactivator CREB binding protein (CBP) -Histone acetylation and chromatin remodelling facilitating transcription.
activators
Eukaryotic organisms are more complex. Thus, they have ________ in addition to the six general transcription factors found in prokaryotes. These proteins bind to enhancers and increase the rate of transcription of a gene in a particular cell type
1. The repressor, attaches to the Operator which prevents the transcription by blocking the RNA polymerase 2. The operon is turned on by the lactose (Which acts as an inducer) by attaching to the repressor 3. The repressor would lose its grip and fall away 4. WHEN THE REPRESSOR FALLS AWAY , THE BINDING OF THE RNA POLYMERASE IS NOW ALLOWED AND THERE FORE THE TRANSLATION IS O DIFFERENT ENZYME WOULD BE PRODUCED
Explain how Inducible Works:
1. The operon will only be turned on when the products are in great demand (in this case arginine) 2. The repressor, isn't able to bind to the operator 3. After some time there would be an accumulation of arg 4. The arginine serves as a co-repressor (meaning it binds to repressor) 5. The Co-represor binds to the represor 6. The co-represor helps bind the repressor to the operator, such that in the presence of an RNA polymerase, the transcription would be stop or prevented
Explain how the Repression Work: Arg operon
Without repressor: The RNA polymerase would bind to the promotor to the start the transcription With Repressor: The repressor binds to the operator and this prevents the RNA polymerase to bind therefore preventing the Transcription
Explain the Negative Regulation
1. Notice that the CRP site is next to the promoter site, 2. when the cAMP CRP is bound it will help push the RNA polymerase from an open form to a closed form 3. The cAMP CRP complex will help the RNA polymerase to clamp down on the DNA particularly on the promoter site and start transcribing the Z,Y,A gene
Explain the Positive Control
1. We have the growth curve in E.coli it grows faster in glucose compared to lactose 2. There is a rapid increase when it is grown in glucose compared when it is grown in lactose 3. When the two sugars are present on a single medium, the glucose will be used first before the lactose 4. In table B, the repressor does not stop transcription 5. The CAP activator, do not bind to the promotor 6. Once the glucose rans out the cyclic-AMP signals that the bacteria is hungry for sugar 7. After signaling the CAP activator will bind to the DNA segment and the lac operon is expressed in the presence of lactose
Explain the table: Lac Operation in Action
During the lag time, the intracellular *Cyclic AMP increases*. So the LAC operon is being transcribed and more lactose is being transported into the cell. That will allow the transport of lactose into the cell. And beta galactosidase is also being synthesized to breakdown that particular lactose.
Explain why the Diauxic growth is happening ?
What is it called when the end product of a metabolic pathway shuts down the pathway by inhibiting the 1st enzyme in the pathway?
Feedback Inhibition
remodeling covalently
For the genes within highly condensed heterochromatin to be expressed, their constituent nucleosomes must undergo ____________ or their constituent histone proteins can be replaced with ___________ modified histone proteins that allow the chromatin to be less condensed.
What gene is not encoded as part of the gal operon and what does this protein do? Why does the gal operon need 2 promoters and what are their purposes?
GalR is encoded distantly from the operon and GalR is the repressor for the gal operon Need 2 promoters because one needs to be active at a baseline level for galactose utilization and transport (pG1) and the other when galactose concentration is high (pG2)
Lactose is made of what two sugars?
Galactose and Glucose
What is happening in each scenario? When galactose is present: Glucose concentration is low -> transcription from promoter ____ Glucose concentration is high -> transcription from promoter _____ When galactose is absent --> __________
Galactose present: Glucose low -> transcription from promoter G1 Glucose high -> transcription from promoter G2 When galactose is absent --> repression by GalR
rRNA
Gene clusters containing repetitive sequences occur for protein coding genes such as those for ________ or tRNA that are required in large quantities
tRNA
Gene clusters containing repetitive sequences occur for protein coding genes such as those for rRNA or _____ that are required in large quantities
What are regulatory genes?
Genes that code for repressors or inducers for the operon that they control. Expressed continuously but at fluxuating levels
Ex. The presence of cAMP (Cyclic AMP) which binds to the catabolic activator protein (CAP) and allows the binding of RNA polymerase on the promoter to start the transcription of the particular gene. Application: In E. coli, when there is the absence of glucose, the lac operon needs an activator (CAP) bound to promoter site for any chance of trascription, regardless of the repressor. In the absence of glucose, the CAP only binds on the operator, in order to allow the RNA polymerase to attach to the promoter. But the presence of glucose the CAP is not required because the organism would favor the utilization of glucose and not the lactose. POSITIVE OPERATOR: lac operon requiring the CAP for the transcription of the gene
Give an example of the Positive Regulation :
How can proteins be activated and example:
Global control (of translation of all mRNAs) involves activation or inactivation of Transcription initiation factors. After fertilization, transcription of mRNA in eggs is triggered by activation of transcription initiation factors.
Histones
Globular protein that assist in DNA packaging in eukaryotes. Histones form octamers around which DNA is wound to form a nucleosome. It binds to DNA's negative charge
Glucocorticoid receptor and cortisol
Glucocorticoid receptor (R) is a transcription factor upon binding of hormone cortisol (A type of glucocorticoid. -Cortisol activates multiple genes -Coordinated gene expression occurs when increased levels of the hormone helps with the transcription of a number of genes -Receptor may be the last missing factor for many genes to transcribe. -A single transcription factor that can be responsible for many processes from cell different ion to organogenesis *transcription factor that can effect multiple genes*
What do we need to make ATP?
Glucose(Sugar)
What bonds a Glucose and a Galactose together?
Glycosidic Linkage
Who are the managers of TFs?
HOX genes
8 uridine residue
Hair pin loop is followed by ? (after ng nakabox, ano ung sunod)
How do HOX TF bind to the DNA gene?
Have an amino acid sequence, a homeodomain. They are coded by homeobox genes which are subsets of HOX genes
High glucose -> ______ cAMP conc -> _____ CAP conc --> _______ expression is activated Low glucose with galactose-> ______ cAMP conc -> _____ CAP conc --> _______ expression is activated and ______ expression is inhibited
High glucose -> low cAMP conc -> low CAP conc --> pG2 expression is activated Low glucose with galactose -> high cAMP conc -> high CAP conc --> pG1 expression is activated and pG2 expression is inhibited
HMG-box motif
High mobility group -3 alpha-helical regions interact with DNA -DNA bending allows for interaction with other transcription factors on nearby sites: architectural factors *SRY Gene*
Under what condition does the ribosome not stall at the trp codons? What does this lead to?
High tryptophan --> no stalling of the ribosome not stall at the trp codons --> ribosome progresses to UGA stop codon at the end of the trpL region and prevents the anti-terminator helix from forming so the terminator helix forms
What helps in the repression of the gal operon by GalR? What is this structure called?
Histone-like protein HU is able to bind between the 2 operators and introduce a 180-degree turn in the DNA that helps to position the operators so that two sets of GalR dimers are able to bind each of the 2 operators and interact with each other to form a stable loop Repressosome
60-80%
How many genes are constitutively produced in a cell?
one
If a hairpin forms between position ______ and position two in the attenuator sequence of the trp operon then transcription will always be prevented
two
If a hairpin forms between position one and position _______ in the attenuator sequence of the trp operon then transcription will always be prevented
transcription terminator
If tryptophan is abundant than the ribosome will proceed quickly along the mRNA strand and will cover position one and position two in the attenuator sequence, allowing formation of the _______ _______
Repressor
Impede access of RNA polymerase to the promotor. -Regulation by a repressor is called negative regulation. -Ligand can either remove repressor protein, or can help in its repression -Binds to OPERATOR
short tandem repeats
In contrast to prokaryotic genomes, which contain essentially all protein coding DNA base pairs, eukaryotic DNA can contain more non-protein coding DNA. In particular, eukaryotic DNA contains repetitive sequences that are referred to as ______ _______ ______ that resulted from template slippage during DNA replication
template slippage
In contrast to prokaryotic genomes, which contain essentially all protein coding DNA base pairs, eukaryotic DNA can contain more non-protein coding DNA. In particular, eukaryotic DNA contains repetitive sequences that are referred to as short tandem repeats that resulted from _______ ________ during DNA replication
Homeodomain
In humans, transcription factors contain a helix-turn-helix domain of 60 amino acids -This is called the homeodomain -Protein that will bind to DNA at recognition helix
Explain the Jacob and Monod model for the regulation of the E.coli lac operon in the presence and absence of lactose? (fig 12.4)
In the absence of lactose: - The repressor (LacI) binds to the operator sequence (lacO) close to the promoter which prevents the binding of RNA polymerase to the promoter and blocks transcription In the presence of lactose: - inducer (allolactose) binds to the repressor and changes its conformation so it can no longer bind to the operator sequence. RNA polymerase can then bind to lacp and transcribe the lac genes
What is the repressor conformation like in the aporepressor state and how does tryptophan binding alter this?
In the aporepressor state, the conformations of the 2 helix turn helix domains in the dimer don't allow proper interactions with successive major grooves - Tryptophan binding alters the HTH conformations to allow the repressor to bind the successive major grooves.
How does AraC self regulate?
In the excess or AraC: AraC binds to araO2 to prevent araC transcription from pc --> self-regulation
Adenine 5'
In the first step of RNA splicing, a specific ____________ within the intron attacks the ___ splice site, cutting the sugar-phosphate backbone of the RNA
TRYPTOPHAN
In the presence of ___________ will enable the binding of the repressor on the operator
What are the structures that form that attenuation transcription and allow the transcript to continue? Where does this occur and what does the formation of these helixes depend on?
In the trpL leader region: Pairing between regions 1 and 2 which create a pause helix and between regions 3 and 4 create the terminator helix which leads to factor independent transcription termination Pairing between regions 2 and 3 creates the antiterminator helix Which helix forms depends on the level of tryptophan present
What is a system that is usually turned off but can be turned on?
Inducible Operon
What type of operon is usually off but can turn on?
Inducible Operon
____________ use the product of a biosynthetic pathway to regulate gene expression
Inducible operons (biosynthetic= making something)
Explain epigenetic inheritance and give an example:
Inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence. (modifications to chromatin that can be reversed.) ex: one identical twin getting schizophrenia, a genetically based disease, while the other twin is fine.
Transcription attenuation
Inhibition of gene expression by premature termination of transcription -Mode of transcription regulation of operons for amino acid biosynthesis. -Upstream of the structure genes in the operon lies a short Open Reading Frame (ORF) for about 14 amino acids. -A relatively large number of the codons in the short ORF code for the amino acid, whose biosynthesis is directed by the operon. -Purpose is to work as an extra brake inhibiting transcription that has escaped the repressor mediated inhibition. *Prokaryotes: Translation and transcription are concurrent*
What protein does regulation of the B. subtilis trp operon involve? How? What determines its shape?
Involves the protein TRAP: trp RNA binding attenuation protein TRAP binds specific 3-base sequences in the leader mRNA only when it has tryptophan bound TRAP is a formally transcriptional repressor, tryptophan is a corepressor Shape determined by binding of tryptophan
For lactose is the repressor protein on or off in the beginning?
It is active, needs a corepressor to inactivate it and for transcription to start.
What happens when a repressor is bound to the operator?
It switches off the operons if in active form (negative control)
Thiogalactoside transacetylase
Lac A codes for
The repressor is produced by what gene? And where is this gene?
Lac L gene, somewhere else on the genome
What is not needed when -Glucose present, less cAMP, less CAP, -Used up lactose, no allolactose, active repressor
Lac Operon
B-galactosidase
Lac Z codes for?
Compare and contrast the lac operon and the trp operon:
Lac operon: catabolic, inducible. Operon codes for enzymes to metabolize lactose, so it's inactive without lactose. Inducer inactivates repressor so genes can be transcribed. Trp Operon: Anabolic, repressible. Operon codes for enzymes to make trp, so its active without trp: repressible. repressor molecule activates repressor protein so genes can function.
How does mutating the regulatory elements of the lac operon (LacI and lacO) alter the system and can they be complemented?
LacI - repressor protein that is made elsewhere in the cell (trans) changing the lacO - site in which the repressor binds means the repressor can no longer bind so RNA polymerase is free to bind.
Co-Repressor: Chromatin remodeling complex
Leads to transcriptional repression -Repressors have subunits with HDAC activity -Co-repressors might also bind histone methyl transferases- COMPACT the chromatin (H3-K9 methylation- Lysine residue #9 of histone H3 methylation) -Hetereochromatic state (Tighten DNA-histone interaction)
Insulator
Limit the action range of enhancers or a repressor -Might bind to nuclear matrix giving looped domains -Barrier sequence prevents spreading of the heterochromatic state of the DNA and allowing for euchromatin state for transcription to be separated. -Limits the enhancer's function to work on a specific site and not jump to another site.
How is looping and contact stabilized?
Looping and contact of activators with other TF (and allows to interact with the Mediator) is stabilized by cohesin, a protein that encircles neighboring strands of DNA
Low glucose -> ___ cAMP -> CAP DNA binding ability ______ High glucose -> ___ cAMP -> CAP DNA binding ability ______
Low glucose -> high cAMP -> CAP able to bind to DNA High glucose -> low cAMP -> CAP not able to bind to DNA
What happens to the aaRS genes in low vs high tRNA aminoacylation concentrations?
Low tRNA aminoacylation: - Absence of an aa on the 3' end of the tRNA allows an interaction with the anti-terminator helix to stabilize it - Sequestration of sequences needed for termination helix formation - Transcription of the aaRS gene High tRNA aminoacylation: - Presence of an aa on the 3' end of the tRNA denies interaction with the anti-terminator helix - Terminator helix forms - No transcription of aaRS gene
Under what condition does the ribosome stall at the trp codons? What does this lead to?
Low tryptophan --> stalling of the ribosome at the trp codons --> region 2 can pair with region 3 and the anti-terminator helix is formed
Mediator
Mediator is a multiprotein complex that functions as a transcriptional coactivator in all eukaryotes. Mediator must also associate with transcription factors and DNA.
hydrophobicity
Methylation increases the __________ of the DNA and as a result increases the stability of the DNA-histone binding.
Why does the chance of cancer increase greatly with age?
Mutations accumulate throughout life, and more than one somatic mutation is needed to produce all the changes of a full-on cancer cell.
No galactose --> GalR ______________ Galactose --> GalR ___________
No Galactose --> GalR represses transcription of the gal operon Galactose --> GalR binds to the galactose inducer and does not repress transcription
No tryptophan --> helix forming between _______ --> results in _________ formation High concentration of tryptophan --> helix forming between _______ --> results in _________ formation
No tryptophan --> helix forming between regions 2 and 3--> results in anti-terminator formation High concentration of tryptophan --> helix forming between regions 1 and 2, regions 3 and 4 --> results in terminator helix formation
If the corepressor isn't present and the repressor protein is what will happen to the operon?
Nothing, because repressor protein isn't activated so it doesn't bind to the activator and turn off the operon. So the operon stays active
Typtophan present, repressor active, operon off or on?
Off
When Tryptophan is present it binds to the repressor protein, which turns that operon off or on?
Off
If Lactose is absent, the repressor is active, is the operon be on or off?
Off, because no corepressor(allolactose) from Lactose
By default the Trp Operon is off or on?
On
Typtophan absent, repressor inactive, operon on or off?
On
The repressor is active only in the presence of its corepressor tryptophan, what does that mean if no tryptophan, for the trp operon on or off?
On-no corepressor
bromodomain TFIID
One of the TAFs within the TFIID general transcription factor contains a __________ the binds acetylated Lys residues on histone tails. This transcription of acetylated residues is increased from the increased availability of the DNA as well as the increased likelihood of the general transcription factor ________ to form and initiate formation of the PIC
What regulation happens in the absence of L-arabinose
P1 AraC preferentially binds to araO2 and araI2 - inhibits binding to araI2 Bends the DNA at pc
What are the two forms of the AraC protein?
P1: Two AraC proteins with no arabinose present --> Antiactivator P2: Two AraC proteins with arabinose bound --> activator protein
trans Golgi network (TGN)
Portion of the Golgi apparatus furthest from the endoplasmic reticulum and from which proteins and lipids leave for lysosomes, secretory vesicles, or the cell surface.
What process ensures that all the tissues and organs of an organism are in their characteristic places? Where do the molecular cues that control this process arise?
Positional information controls pattern formation, development of front, back, top, bottom, and sides of embryo. Positional info is provided by cytoplasmic determinants and inductive signals. Tells the cell its relation to other cells & the body axes. Determines how the cell & its progeny will respond to future molecular signals.
The E. coli ara operon is an example of what kind of regulation operon?
Positive regulation
Attenuation
Premature termination of transcription
What regulation happens in the presence of tryptophan?
Presence of tryptophan: The trpR gene is transcribed as an aporepressor. Tryptophan (the corepressor) binds to TrpR to make an active repressor which can bind as a dimer to the promoter to repress transcription of the Trp operon
In the what type of cell are genes arranged adjacently?
Prokaryotic Cells
Tryptophan operon
Promoter sequence and 5 genes -Tryptophan biosynthesis -Inactive repressor protein not bound to operator (not repressing Trp genes) -When Trp becomes abundant it will act as a co-repressor by binding to the repressor protein, which becomes activated to bind to the operator. -Repressor protein is always present in 20 copies per ell (constitutive expression)
Protein Kinases: Initiation of translation
Protein Kinase can influence the initiation of translation Example: -Globin synthesis in reticulocytes is dependent on phosphorylation of eIF2 (eukaryotic initiation factor 2) -Heme is reversible inhibitor of kinase; Heme activates eIF2, while kinase makes eIF2 unstable. *Active eIF2: When iron is available then hem is present in the reticulocyte and the kinase is inhibited. *Unstable: Iron is not available, then there will be no heme; this would lead to kinase phosphorylating eIF2, leading to its degradation *Kinase phosphorylation allows to regulate eukaryotic translation*
gene-specific transcription factors
Proteins that bind to enhancers or silencers of DNA, i.e. regulatory sequences
What is a group of control elements near the promoter called?
Proximal Control Elements
What is a riboswitch? What is everything dependent on?
RNA element that is usually found in leader RNAs that controls expression of the downstream coding sequences by sensing a regulatory signal directly - doesn't need a regulatory protein - senses ligand directly Everything depends on folding
phosphorylated
RNA pol is __________, causing a conformation change. It is then released from the promoter to begin transcription
How can factor independent termination occur for transcription attenuation regulated genes?
RNA polymerase pausing during U synthesis allows nascent RNA emerging from the RNA pol to fold into the terminator helix Triggers RNA polymerase to terminate transcription -->> factor independent termination
-5' capping -3' polyadenylation -exon splicing
RNA processing involves:
spliceosome
RNA splicing is performed by a complex of small nuclear RNA (snRNAs) and ribonucleoproteins called:
nuclear pore complexes
RNAs exit via _______ _____ ________ that cover the nuclear membrane
Helix-Loop-Helix
Region of 50 amino acids that are involved in protein dimer formation. -DNA binding is mediated by an adjacent short stretch of basic amino acids (bHLH) -MyoD: Transcription factor involved in muscle differentiation contains a bHLH domain.
Gene Expression
Regulated gene expression affect the production of product: Induced or repressed genes. *Constitutive Gene Expression: Housekeeping genes that are kept constant more or less
What is the function of TrpR and where is its gene located relative to the operon?
Regulates the trp operon and aroH operon (for chorismic acid synthesis) Gene is unlinked from the rest of the operon
What are the two main ways of controlling metabolism in bacterial cells?
Regulation of Enzyme activity: feedback inhibition Regulation of enzyme production: repress expression of the genes that code for the enzymes in this pathway.
What is post-transcriptional regulation? What type is the most common?
Regulation that occurs after the gene is transcribed into mRNA - most common is translational regulation
What type of operon is usually on but can turn off?
Repressible Operon
What occurs when there is an interaction between the repressor protein and the 2 operators?
Repressor molecules bound to the 2 operators interact with each other to bend the DNA of the promoter that is between the 2 operators Bent promoter does not bind RNA polymerase so there is no initiation of transcription at the promoter
What happens when tryptophan concentration drops?
Repressor protein doesn't bind to the activator and stop the transcription, so we make lots of enzymes
How do repressors do their job?
Repressors act by directly interfering with activator binding with silencers that indirectly interfere with activators
What kind of DNA does regulation of the gal operon usually require and why?
Requires supercoiled DNA presumably for accurate placement of the GalR dimers to form the DNA loop
SRY gene
Responsible for male sexual differentiation -Contain a HMG box -Protein SRY bends DNA -Protein SRY from Y-chromosomal SRY gene: Involved in testis differentiation -Mutated SRY= Sex reversal
What is the trp operon responsible for?
Responsible for synthesizing the amino acid L-tryptophan for proteins - needs to be made if it is not in the media as chorismic acid
Deficiency of HATs
Results in complex Genetic Disorders *Rubinstein-Tabyi Syndrome*: Postnatal growth deficiency, microcephaly, specific facial characteristics, broad thumbs, big toes, and mental retardation. -Mutations in the CBP protein or in the closely related p300 protein who both have HAT activity.
Iron-responsive elements
Secondary mRNA structures (hairpins) can get involved in translation regulation -Ferritin is an iron-storage protein required when there is an excess of dietary iron; iron storage protein -Transferrin receptor is involved in the uptake or transferrin-bound iron by cells (important for iron deficiency); iron uptake protein. -Iron Responsive Element (IRE) regulates ferritin and transferrin receptor.
2
Sequence 1 is partially complementary to sequence _____________
3
Sequence 2 is partially complementary to sequence __________
What does it mean when its said that the galactose utilization pathway is an Amphibolic pathway? Why is the gal operon expressed at a minimal level at all times?
Serves both a catabolic and anabolic purpose Cell needs the genes from the gal operon to make LPS via glycosylations
Explain the different inductive signals for how different sets of activators come to be present in two cells:
Signals to the embryonic cell from other embryonic cells in the vicinity. ex: binding of growth factors secreted by neighbouring cells. These signals cause changes in the target cell (induction) and send the cell down a noticeably differentiated path.
Specificity Sigma Factors
Sigma subunit 70 binds to RNA polymerases to become a holoenzyme that binds to the promoter sequence. -During certain conditions (like heat): The Sigma subunit 32 will replace the sigma subunit 70. -With a different sigma factor, RNA polymerase is directed to a specialized polymerase to a specialized set of promoters. -Can induce the production of heat shock proteins *Sigma factor can change depending on the condition, leading to regulation of genes*
What are RNA thermosensors? What happens when temperatures rise?
Some RNA secondary structures block ribosome access to the TIR of the mRNA (SD and/or initiation codon) - when temperatures rise the secondary structures melt
Other HOX genes mentioned
Sox POU Lim T-Box
Glucocorticoid: Transcriptional Regulation
Steroid hormone with the ability to cross membrane, and bind to HRE in target genes -Glucocorticoid will cross the PM via diffusion -HSP 90 protein is bound to two inactive glucocorticoid receptors that are INACTIVE -After glucocorticoid crosses, it will interact with HSP90 via binding to R site. -R site is released from HSP90 and will form dimerization with the other R (RR complex) -RR is now has a nuclear localization signal- causes RR to move to the nucleus -RR dimer activate genes with the HRE in their promotor via chromatin remodeling coactivators.
Competition among the number of activators and repressors provides?
Subtle regulation and flexibility in both the direction (activate, suppress) and the overall degree of gene expression.
What happens when you take a cell with a RNA thermosensor and do the following: - start growth at 20 degrees then switch to 40 - start growth at 40 and switch to 40 - start growth at 20 degrees and increase the temperature slowly in 5 degree increments until you reach 40 --> what does this allow for?
Switching the growth temp from 20 to 40 degrees quickly -> will not grow Then going from 40 to 40 -> will grow normally Switching the temperature by increments 20 - 25 - 30 - 35 - 40 -> the cells will grow -> Induction process, the cell needs to adjust to the new temperature and allow folding for the cells to be able to grow
HOX genes produce
TF that generate the body plan and organ development
transcription factors for RNA polymerase II
TFII
TFIIA
TFIIE and TFIIH join to form the preinitiation complex. (this is a form of general transcription)
development
TFs are critical for:
complexes
TFs can assemble as:
homomeric heteromeric
TFs can form _______ and _______ proteins (dimers, trimers, tetramers, etc)
repressors
TFs that bind DNA, turning genes off are called ________ or gene repressor proteins
activators
TFs that bind DNA, turning genes on are called _____ or gene activator proteins
FALSE: closed form (ung nasa picture)
TRUE OR FALSE: RNA polymerase in Open form can transcribe genes
FALSE: Repression works when there is an excess co-repressor which would bind to the Repressor
TRUE OR FALSE: Respression only works when there are a few co-repressors
False:Not all genes are expressed at any one time, nor are they always expressed at the same level
TRUE OR FALSE:All genes are expressed at any one time, and they are always at the same level
small large
ribosomes are composed of 1 ________ and 1 _______ large subunit
What are the 2 possibilities that the RNA secondary structure might lead to for transcription attenutation?
Terminator: RNA secondary structure that triggers factor-independent transcription termination Antiterminator: RNA secondary structure that captures sequences necessary for terminator helix formation
Explain what occurs in morphogenesis:
Th physical processes that give an organism its shape. Literally meaning, "creation of form." Changes in shape, motility, and other characteristics of the cells that make up sections of the embryo.
The E. coli tryptophan operon is a ________ repressible system
The E. coli tryptophan operon is a negative repressible system
1. Lac z, Lac Y, Lac A 2. Promoter
The Lac Operon's structural gene is composed of?
promoter
The RNA polymerase binding site of the DNA is referred to as a _________
Repressor
The Regulatory gene of the Lac Operon is made up of
membrane-bound
ribosomes in co-translational transport
Lac Repressor binds to what to block transcription?
The activator on the operon
What does it mean when its said that araC exhibits face of the helix dependence?
The binding site for a regulatory protein on the regulated gene is constrained by how a regulatory protein bound to that site will be positioned relative to some other site
CAP
The cAMP-______ complex simulates transcription by binding the promoter
Why are there 2 promoters in the gal operon?
The cell always needs galactose
genes size
The complexity of an organism's morphology and metabolism increases with the number of ________ in its genome, but not the ______ of that genome (the number of constituent base pairs)
genes base pairs
The complexity of an organism, in terms of the complexity of its morphology and metabolism, increases with the number of ______ in the genome of the organism, but does not increase with the number of ______ ______ of that genome
reversible
The four types of post transcriptional modifications that occur to histones to alter DNA transcription as a function of the density of the chromatin are ubiqitination, phosphorylation, methylation, and acetylation. These reactions are readily __________ by certain enzymes
What is the state of the functional TrpR respressor like? What are the critical structures?
The functional TrpR repressor is a dimer of two TrpR monomers Each monomer has helical structures that are critical for HTH region for DNA binding
CAP
The increased concentration of camp increases transcription of the lac operon because camp binds the _____ (acronym) protein, which then binds the promoter of the lac operon and stimulates transcription in the absence of the lac repressor
transglycosylation
The inducer for the lac repressor protein, allolactase,, which indirectly provides a signal for high lactose concentrations, is produced from a ______________ reaction of lactose.
1. Sugar lactose 2. A regulatory gene 3. 2 control elements
The lac operon is involved in the processing of:
Figure 12.6: The lac operon regulatory region: What kind of promoter is the lac promoter? What does the RNA polymerase interaction region include? Where are the operator sites and to which does LacI bind?
The lac promoter is a typical σ70 promoter RNA polymerase interaction region includes the -35 and -10 region and the repressor binding site (operator 1) O2 and O3 are positioned upstream and downstream of the promoter LacI binds to O1 and by binding inhibits RNA polymerase from binding
allolactase
The lac represor protein is prevented from binding to the operator by an inducer. The inducer for the lac repressor protein is _________
operator promoter
The lac repressor protein binds the ________ to prevent the action of rna polymerase whereas the CAMP-CAP complex binds the ________ to stimulate transcription
4
The leader transcript involves how many short sequences?
The ____ of the two operator sites relative to each other is critical for regulation.
The position of the two operator sites relative to each other is critical for regulation.
what is differentiation?
The process by which a cell or group of cells become specialized in structure and function
What determines the specificity of the tRNA antiterminator interaction?
The specifier loop : unpaired region that includes a triplet sequence corresponding to the aa specificity of the regulated aaRS binds to the anticodon
corepressor
The trp repressor protein, in complex with tryptophan, specially binds to the trump operator to prevent transcription of the trp operon. In this mechanism, the tryptophan acts as a __________ to prevent superfluous tryptophan production
repulsion
There are four types of post transcriptional modifications that occur to histones: acetylation, phosphorylation, methylation, and ubiquitination. All of these post transcriptional modifications increase the negative charge of the histones and thereby causes the chromatin to decondense through change ___________
What are egg polarity genes?
This is a different name for maternal effect genes, since they also set up the axes of an embryo.
3-4 structure
This struture is the Transcription termination sequence
How can mRNA that is transcribed be regulated?
Through translational regulation where translation is inhibited
chromic remodeling complexes
To decreases the density of heterochromatin and make the DNA available for transcription, interactions between the nucleosomes and the DNA is disrupted by dependent _________ _________ ________ that facilitate ATP dependent reactions that unwind the DNA from the nucleosomes.
ATP
To decreases the density of heterochromatin and make the DNA available for transcription, interactions between the nucleosomes and the DNA is disrupted by dependent chromatin remodeling complexes that facilitate ________ dependent reactions that unwind the DNA from the nucleosomes.
What is the role played by noncoding RNAs?
To regulate gene expression. Regulation by noncoding RNAs occurs at mRNA translation & chromatin configuration.
What is the purpose of the E. coli trp operon? What are the 2 levels it is regulated at?
To synthesize tryptophan -> if tryptophan is present, expression of these genes is unnecessary The operon is regulated at 2 levels: 1. Transcription initiation 2. Transcription attenuation
Explain how S-adenosylmethionine (SAM) is an example of a metabolite-binding riboswitch in B. subtilis. What does this molecule help the cell determine?
Too much SAM: have an anti-antiterminator forming à inhibits the antiterminator so makes it terminate -At this point will determine if more methionine is needed by sensing the presence of SAM The cell determines its need for methionine biosynthesis by monitoring the level of SAM
To initiate transcription, RNA pol II needs assistance of _______________.
Trancription factor proteins.
What is the most common level of gene expression?
Transcription
What is the most energetically favorable(less waste) level of gene expression?
Transcription
The TATA Box is an example of what?
Transcription Factor
In scenario: Lactose present, little glucose, (high cAMP levels), active CAP by cAMP bind to CAP site, which acts as what?
Transcription Factors
What are proteins that interact with DNA and affect the transcription?
Transcription Factors
What contains the following domains? -DNA binding domain -activation domain
Transcription Factors
What is transcription attenutation and what structure impacts whether this occurs or not?
Transcription begins constitutively at the promoter but is terminated in the leader region of the transcript before RNA polymerase reaches the first structural gene The secondary structure of the RNA impacts whether its transcription continues or is terminated
RNA polymerase (Promotor)
Transcription depends not only on the operator but also an attachment sites of _______________
Where are TF's located?
Transcription factors TF are located throughout the cytoplasm and the nucleus
Leucine Zipper (heterodimers)
Transcription factors is enhanced by formation of heterodimers. -Heterodimers allows for 2 different domains to recognize different promoter sequences, thus allowing for variation in products made.
What genes are t-RNA-sensing riboswitches in B. subtilis involved in regulating? What does depriving the cell of this lead to?
Transcription of genes encoding aminoacyl-tRNA synthetase --> the enzyme that charges the tRNA with an amino acid Depriving the cell of aaRS causes accumulation of uncharges form of tRNA - bacteria can synthesize higher levels of the aaRS for that particular tRNA - more efficient attachment of the limiting aa to tRNA
How does an activator know where to attach to the DNA? In other words, how do proteins know how to bind to nucleic acid bases?
Transcription regulator/factor recognizes a DNA sequence because the surface of the protein is COMPLEMENTARY to specific features that include patterns of hydrogen bonds, ionic bonds and hydrophobic interactions among the base pairs
What is the most common and efficient type of regulation? Can all genes be regulated this way?
Transcriptional regulation is the most common and efficient type of regulation. But not all genes are regulated at the transcriptional level.
Tamoxifen
Treatment of breast cancer -Breast tumors-> Rely on estrogen for growth -Tamoxifen is an estrogen analog and binds to pockets estrogen would bind to (competitive antagonist) -Tamoxifen binding distorts the struct of estrogen receptor -Coactivator will not bind (estrogen NOT there), but instead co-repressor will bind and block estrogen defects leading to no cell division.
False: Other Genes are not expressed all the time, they are switched on and off depending on the need of organism
True or false: All genes are expressed all the time, they are always switched on
B. subtilis trp operon: How does transcriptional attenuation regulate the operon in the presence or absence of tryptophan?
Tryptophan not present: - TRAP cannot bind - anti-termination helix is formed Tryptophan present: - TRAP is able to bind -Termination helix is formed
B. subtilis trp operon: Explain the translational control when tryptophan is present vs when it isn't.
Tryptophan present: - TRAP binding close to the Shine Dalgarno sequence prevents the ribosome from binding and blocks translation - Sequesters the Shine Dalgarno sequence Tryptophan not present: - no TRAP binding - SD is available to the ribosome
Tryptophan and Attenuation
Tryptophan reduces expression by a factor 70 while attenuation can decrease expression by a factor of 10. -Trp binds to repressor, allowing it to bind to operator; then with high tryptophan levels, attenuation will help with decreasing tryptophan levels -Cellular Trp + Attenuation= repression of trp operon by 700 fold
What are the types of transcriptional regulation? Explain both.
Types of transcriptional regulation: - Regulation of transcriptional initiation: regulation occurs at the operator/promoter site for transcription to begin or not - Regulation of transcriptional attenuation: regulation occurs after the RNA polymerase leaves the promoter
lactose
Under normal conditions when __________ concentrations are low, transcription of the lac operon is prevented by the lac repressor protein that binds the operator sequence and blocks RNAP from transcribing the operon.
repressor operator
Under normal conditions when glucose is available, bacteria do not synthesize the enzymes for the metabolism of lactose. Transcription of the lac operon under low glucose conditions is prevented by the lac _________ protein, which binds to the ________ sequence downstream of the promoter to prevent transcription of the structural genes by the RNAP holoenzyme
promoter
Under normal conditions when glucose is available, bacteria do not synthesize the enzymes for the metabolism of lactose. Transcription of the lac operon under low glucose conditions is prevented by the lac repressor protein, which binds to the operator sequence downstream of the ________ to prevent transcription of the structural genes by the RNAP holoenzyme
Promotor Sequence
Variations in promotor sequences can account for 1000 fold difference in mRNA product -Determinate for expression of house keeping gene -Deviation from consensus sequence= reduced promotor function
These are the genes that are involved in the vital biochemical processes such as respiration
What are the Genes that are needed to be expressed all the time
1. Structural genes 2. Regulatory gene 3. Operator 4. Inducer
What are the components of a Lactose Operon is made up of ?
1. Tryptophan E 2. Tryptophan D 3. Tryptophan C 4. Tryptophan B 5. Tryptophan A (ED Caluag BA???)
What are the five structural genes which codes for tryptophan synthetase
1-2, 2-3, 3-4
What are the three distinct secondary structure (hairpins) can be formed from the complementary sequence?
B-galactosidase, Lactose permease, Thiogalactoside transacetylase
What are the three genes that lac Operon codes
1. Initiation 2. Elongation 3. Termination
What are the three steps of transcription?
(1) Trytophan repressor/ initiation (2) attenuation
What are the two layer of regulation involved
1. Negative Transcriptional System 2. Positive Transcriptional System
What are the two types of Transitional Control System
-eIF-2B can't function in GTP exchange -eIF2 remains inactive -protein synthesis is slowed
What happens if eIF2 is phosphorylated?
Lactose
What is the inducer of lac operon ?
If tryptophan levels in the cell are high, the ribosome will translate the entire leader peptide without interruption and will only stall during translation termination at the stop codon. At this point the ribosome physically shields both sequences 1 and 2. Sequences 3 and 4 are thus free to form the 3-4 structure which terminates transcription of the succeeding part of the operon hence prevents the formation of proteins.
What will happen if the Tryptophan level is high?
tryptophan levels in the cell are low, it will stall at either of the two trp codons. While it is stalled, the ribosome physically shields sequence 1 of the transcript, thus preventing it from forming the 1-2 secondary structure. Sequence 2 is then free to hybridize with sequence 3 to form the 2-3 structure, which then prevents the formation of the 3-4 termination hairpin, thus the 2-3 structure is called anti-termination hairpin. RNA polymerase is free to continue transcribing the entire operon
What will happen if the tryptophan level is low?
Allows gene expression
What will happen to transcription in the absence of tryptophan
No gene expression and Activates repressor
What will happen to transcription in the presence of tryptophan
When the tryptophan concentration is low, the Trp repressor is free of its co-repressor and vacates its operator, allowing the synthesis of trp mRNA to commence from the adjacent promoter
What would happen if the tryptophan concentration is low?
1. Inactivation of repressor (because the lactose acts as inducer that binds to the repressor causing it to fall off from the gene) 2. Expression
What would happen if there is a presence of lactose
1. There is an active repressor 2. No expression of the gene
What would happen if there is absence of lactose?
Becaus glucose is absent the cyclic AMP is present in high amount so the cyclic CRP complex is bound but cannot stimulate transcription because RNA polymerase because of the repressor *No transcription*
What would happened to the LAC operon when Glucose and Lactose are absent ?
The cyclicAMP-CRP compex is bound on to the gene. At the same time the lactose binds to the repressor so it falls off. The Z,Y,A genes are transcribed in large amounts because the cAMP-CRP complex allows the clumping of RNA polymerase on the gene and there would be full transcription on the gene. *OPERON IS TURNED ON FULLY , transcription*
What would happened to the LAC operon when Glucose is absent and Lactose is present ?
In this case the cyclic AMP is absent (no lactose) there is NO transcription. Because the repressor is still present that is bound to the operator and there are no cAMP-CRP complex to allow the campling of RNA polymerase to the gene. OPERON IS OFF *no transcription*
What would happened to the LAC operon when Glucose is present and Lactose is absent ?
If lactose and Glucose is present, since glucose is present the Cyclic AMP is absent. So the Z,Y,A genes are very weakly transcribed. So generally the lac operon is opt because the CAP is not bound to CRP. SO THERE WOULD BE MINIMAL TRANSCRIPTION, AND THE LACTOSE REPRESSOR HAS FALLEN OFF BUT THE cAMP-CRP complex is not bound so it could not stimulate transcription the transciption of operon at a high level. In this case the lactose is present, suppostedly it will bind to repressor to allow the RNA polymerase to transcribe the gene, but although the repressor in particular falls off because of the presence of lactose, the glucose the cAMP-CRP complex is not bound onto the promotor hence the *operon is OFF and there is small amount of transcription happens*
What would happened to the LAC operon when Lactose and Glucose are present
What regulation happens in the presence of L-arabinose
When arabinose is bound, two AraC monomers bind araI1 and araI2 to activate transcription from pBAD
When is CAP used?
When glucose levels are low
camp
When glucose stores are low, transcription of the lac operon is increased. This results from an increase in _______, which indirectly is a signal that glucose is low.
Deletion Map
When looking at Map: *High percentage: The sequence removed must have been a repressor, as the relative promoter activity has increased. *Low percentage: The sequence removed must have been an activator, as the relative promoter activity has decreased.
What is DNA methylation? How does it affect gene expression?
When methyl groups are added to certain bases, that section of DNA becomes inactive. Removal of the methyl can allow transcription to occur: there are proteins that bind to methylated DNA & recruit enzymes to demethylate them.
-promotes cleavage of the mRNA -inhibits protein translation of the target mRNA
When miRNA binds specific sequences in target mRNA, two things can happen:
free in cytosol
ribosomes in post-translational transport
What happens when a ligand is bound to the riboswitch? When a ligand isn't bound?
When the ligand is bound: - a terminator helix is formed - transcription is terminated When there is no ligand bound: - an anti-terminator helix is formed --> U's are far from the hairpin loop - transcription is not terminated
The transcription is prevented (like a traffic light that acts as a go or stop signal for transcription of the structural genes)
When the operator is bound to repressor, what happened to the transcription?
Trp repressor
When tryptophan is present, it binds this repressor and induces a conformational change in that protein, enabling it to bind the trp operator and prevent transcription
What is genomic imprinting, and how is it maintained?
Where methylation permanently regulates expression of either the maternal or paternal allele of particular genes at the start of development. (X chromosome methylation)
attenuator
Within the leader sequence of the trp operon is an _______ sequence, which contains four positions capable of forming hairpin structures and two tryptophan codons located between positions one and two.
tryptophan
Within the leader sequence of the trp operon is an attenuator sequence, which contains four positions capable of forming hairpin structures and two ________ codons located between positions one and two.
Is lactose a dissacharide?
Yes, Galactose and Glucose
mediator
a _________ is a protein that facilitates the interaction between the activators bound to enhancers and the RNAP bound to the promoter.
bromodomain
a ___________ is region of the protein that binds Lys residues acetylated histone tails
bromodomain chromodomain
a ____________ binds acetylated lysine residues in histone tails whereas a ___________ binds methylated residues in histone tails
when lactose is present
a bacteria will turn on genes in the lac operon order to digest the lactose
post-transcriptional regulation , after an mRNA is produced
a cell has mechanisms to control the amount of protein produced
Alarmolne
a chemical alarm signal that promotes a cell's response to the environment or nutritional stress (ex. cAMP- when cell doesnt have glucose it makes a cyclic AMP which is a signals the cAMP receptor protein that a cell is hungry for sugar)
corepressor
a compound that binds a repressor to increases it's binding to the operator is referred to as a ____________
consensus sequence
a conserved nucleotide sequence of DNA, RNA or amino acid sequence that is generally used for molecular interactions
proximal control elements
a control element that is located close to the promoter
bromodomain
a domain on a protein that binds acetylated lysine residues on histone tails is referred to as a ___________
regulatory gene
a gene that codes for a protein, such as a repressor, that controls the transcription of another gene or group of genes
proteasome
a giant protein complex that recognizes and destroys proteins tagged for elimination by the small protein ubiquitin
Operon
a group of functionally related genes, close to each other that are transcribed at the same time
antiterminator
a hairpin formed between positions two and three in the trp attenuator sequence is referred to as an _________
Feedback inibition
a method of metabolic control that prevents synthesis of enzymes that are not needed , this prevents waste energy as well as the wastage of materials of the cell
Feedback inhibition
a method of metabolic control that stops the enzyme that have already synthesized
endoplasmic reticulum (ER)
a network of membrane enclosed tubules and sacs (cisternae) extending from the nuclear membrane throughout the cytoplasm
genomic imprinting
a phenomenon in which expression of an allele in offspring depends on whether the allele is inherited from the male or female parent
attenuation of HIV viral DNA
a protective mechanism for the human genome
catabolite activator protein (CAP)
a protein inducer that binds to the DNA in the presence of lactose and no glucose allowing transcription of lac proteins
activator
a protein that binds to DNA and stimulates gene transcription. in prokaryotes, activators bind in or near the promoter; in eukaryotes, activators bind to control elements in enhancers
repressor
a protein that inhibits gene transcription; in prokaryotes, they bind to the DNA in or near the promoter; in eukaryotes, they may bind to control elements within enhancers, to activators, or to other proteins in a way that blocks activators from binding to DNA
Activator Protein (Expressor)
a regulatory protein that enables RNA polymerase to begin transcription
histone
a remodeling complex is a protein complex that disrupts the interactions between _______ proteins and DNA
DNA
a remodeling complex is a protein complex that disrupts the interactions between histone proteins and ______
enhancer
a segment of eukaryotic DNA containing multiple control elements, usually located far from the gene whose transcription it regulates
control elements
a segment of noncoding DNA that helps regulate transcription of a gene by binding a transcription factor. multiple control elements are present in a eukaryotic gene's enhancer
what is the operator?
a sequence of DNA that controls access of RNA pol to a cluster of functionally related genes. Positioned within promoter or between promoter & genes
What is a promoter?
a site where RNA pol can bind to DNA to begin transcription
corepressor
a small molecule that binds to a bacterial repressor protein and changes its shape, allowing it to switch an operon off
ubiquitin
a small regulatory protein found in most tissues
small interfering RNA (siRNA)
a small, single-stranded RNA molecule generated by cellular machinery from a long, double stranded RNA molecule; associates with one or more proteins in a complex that can degrade or prevent translation of an mRNA with a complementary sequence; can block transcription in some cases by promoting chromatin modification
microRNA (miRNA)
a small, single-stranded RNA molecule, generated from a hairpin structure on a precursor RNA transcribed from a particular gene; associates with one or more proteins in a complex that can degrade or prevent translation of an mRNA with a complementary sequence
Each HOX gene is a transcription factor for?
a specific part of the body; PAX6 - development of eye
inducer
a specific small molecule that binds to a bacterial repressor protein and changes the repressor's shape so that it cannot bind to an operator, thus switching an operon on
RNA interference (RNAi)
a technique used to silence the expression of selected genes; uses synthetic double stranded RNA molecules that match the sequence of a particular gene to trigger the breakdown of the gene's messenger RNA
alternative RNA splicing
a type of eukaryotic gene regulation at the RNA-processing level in which different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as intron
Negative Transcriptional Control System
a type of transcriptional Control system that is either *inducible or repressible*
Positive Transcriptional Control System
a type of transcriptional control system that codes for a protein necessary for the gene to be expressed (Codes for an activator protein)
operon
a unit of genetic function found in bacteria and phages, consisting of a promoter, an operator, and a coordinately regulated cluster of genes whose products function in a common pathway
disease
abnormal alternative splicing can cause ________ i.e. abnormal processing of the b-globin transcript in b-thalassemia
What does the p53 tumor suppressor gene do?
activated p53 promotes transcription of the gene for a protein that inhibits the cell cycle, ensuring that damaged DNA cells aren't replicated. Or, it just blocks the cells cycle until the damage can be repaired. When DNA damage is irreparable, p53 activates genes whose products cause apoptosis.
CRP is an
activator of transcription
By itself, the lac repressor is ___ and switches the lac operon __
active, off
DNA methylation is the
addition of methyl groups to certain bases in DNA
5' capping
addition of methyl-guanosine to 5' end
protein glycosylation
addition of oligosaccharides to proteins; occurs in golgi
3' polyadenylation
addition of polyA tail at 3' end
some target genes for MyoD (protein) encode
additional muscle-specific transcription factors
How are the actions of repressors and activators taken on a single gene?
additively
genome
all cells have the same _______, the same set of DNA instructions
structure of insulin
alpha chain and beta chain connected by disulfide bonds
regulated
alternative splicing can also be ________ by activator and repressor proteins
housekeeping genes
always on i.e. metabolism proteins, DNA polymerases
What senses the tryptophan? What does this molecule need it for?
aminoacylated tRNAtrp --> needs the tryptophan to charge the tRNA
activation domain
an _______ ________ is an acidic region found in upstream transcription factors
operon
an _______ is a cluster of functionally related genes that are regulated by the same promoter.
enhancer
an _______ is an activator binding site
enhanceosome
an ________ is a complex of proteins containing coactivators or corepressers that act in concert to regulate transcription in eukaryotes
activator
an ________ is a protein that binds an enhancer sequence
insulator
an _________ is a region of DNA that defines the range of interaction between the enhancer and the promoter
attenuator
an __________ is a transcription terminator that is present in certain operons
corepressors
an enhanceosome is a complex of proteins containing coactivators or _________ that act in concert to regulate transcription in eukaryotes
homeotic gene
any of the master regulatory genes that control placement and spatial organization of body parts in animals, plants, and fungi by controlling the developmental fate of groups of cells
25,000 60
approximately _________ genes in the human genome; estimated that __% genes undergo alternative splicing
in histone acetylation, acetyl groups
are attached to an amino acid in histone tails
What is the common control point of gene expression for all organisms?
at transcription: regulation at this stage is often in response to signals coming from outside the cell (ex:hormones). For this reason, gene expression is often equated with transcription.
Explain why CAP binding and stimulation of gene expression is positive regulation:
because CAP binds directly to the promoter so it directly stimulates gene expression
competition
because mRNA 5' cap and 3' polyA tail are used in both translation initiation and deadenylation-dependent mRNA decay, there is ________ between mRNA translation and mRNA decay
RNA polymerase II
before transcription can begin, ________ ______ ___ requires general transcription factors to assemble at the promoter
Lac Z, because beta galactosidase hydrolyses the lactose into a molecule of glucose and the molecule of galactose
between Lac Z, Y, A which one is the most important
the trpR gene codes for a repressor protein that will
bind to the trp operator and block transcription of the trpE to trpA genes
chromatin
blocks access of TF's to potential DNA-binding sites
How can both repressible and inducible operons be negative regulators?
both need inducer/repressor proteins to bind to the regulatory protein in order to function.
when is RNA polymerase released?
by control of the transcription factors. (released from GTP)
How is the Lac Repressor controlled?
by the corepressor Allolactose
Describe the relationship between the glucose supply, cAMP, and CAP.
cAMP accumulates when glucose is scarce. When cAMP binds to CAP, CAP becomes active and binds to an upstream area of the lac promoter. This increases the affinity of RNA pol for the promoter. CAP facilitates RNA pol binding and increased rate of transcription. If glucose is in high concentration and cAMP is low, CAP detaches from the operon. Then RNA pol binds less efficiently and rate of transcription is decreased.
cAMP binds to the
cAMP receptor protein (CRP)
gene expression
can be different in normal cells vs a diseased cell
Where can distal control elements be?
can be in an intron, can be grouped as enhancers or separate, and can be near or far from the promoter.
specialized genes
can either be on or off i.e. Hb in RBCs
In response to chemical signals, prokaryotes can do what?
can turn off translation of their mRNA
How does a repressor protein work?
can turn on/off an operon. binds to operator & blocks attachment of RNA pol to promoter
What is CAP? how does CAP work?
catabolite activator protein is a regulatory protein that binds to DNA & stimulates transcription of a gene. Positive regulator.
GTP hydrolysis
causes eIFs to dissociate, allowing large ribosomal subunit to bind, and begin protein synthesis
histone methylation
causes nucleosomes to pack tightly together which inactivates DNA transcription, so genes are not expressed
ATP hydrolysis
causes the protein complex to push on the DNA and "loosen" its attachment to the histone core
miRNAs can inhibit protein translation by
causing the degradation of the mRNA or preventing translation at the ribosome
What are the three processes that lead to the transformation of a zygote into an organism?
cell division, cell differentiation, and morphogenesis.
exosomes
cell-derived secreted vesicles that regulate target cell gene expression and activity
Explain what occurs in cell differentiation:
cells become specialized in structure and function, and these are organized into tissues and organs. Activities of a cell depend on the genes it expresses and the proteins it produces. Specific activators turn on the collection of genes whose products are needed in the cell. Materials placed into the cell by the mother set up a sequential program of gene regulation that's carried out as cells divide.
what are myoblasts?
cells determined to form muscle cells and produce large amounts of muscle-specific proteins
communication between cells is essential for
cells to become different cell types
chromatin structure
changes in ________ ______ play a major role in regulating gene expression
Bacterial operons
cluster of functionally related genes can be coordinately controlled by a single "on-off" switch
lac Z
codes for β-galactosidase for lactose hydrolysis
v-SNARE t-SNARE
complementary ______ and ___________ proteins form stable complexes, thereby bringing vesicle and the target membrane bilayers into close apposition for fusion
anticodon
complementary base pairing with mRNA occurs here
1. specific DNA sequences 2. proteins that bind them
components of genetic switch
the lac operon contains
genes that code for enzymes used in the hydrolysis and metabolism of lactose
the amount of RNA or protein stays at a fairly ______ level in the cell
constant
Repressible
contain genes coding for anabolic enzymes; several genes in a series are turned off by the product synthesized by the enzyme
What would occur if the repressor of an inducible operon were mutated so that it couldn't bind to the operon?
continuous transcription of the operon's genes
Inducible genes
genes whose expression is turned on by the presence of some substance
How are TFs activated and regulated?
coregulators
A lack of this nonprotein molecule would result in the inability of the cell to 'turn off' genes:
corepressor (nonprotein molecule)
when glucose is scarce,
cyclic AMP (cAMP) levels rise
cyclic amp (cAMP)
cyclic adenosine monophosphate, a ring-shaped molecule made from ATP that is a common intracellular signaling molecule (second messenger) in eukaryotic cells. it is also a regulator of some bacterial operons
Zinc fingers
cysteine and histidine residues bind to a Zn2+ ion, looping the amino acid into a finger-like chain that will rest in the grooves of DNA
E site
deacylated tRNA exit via the:
phosphatases
dephosphorylate
(eukTR) a complex mix of activators and repressors
determines what genes are turned on or off in different cell types
Using the metabolic needs of e. coli, explain why all genes aren't on all of the time:
e. coli living in the stomach of a human have to adapt to whatever the person eats, and they also need tryptophan for their own fuel. E. coli activate their own metabolic pathway to make trp in absence, but if the person eats it the presence inhibits enzyme 1 by feedback inhibition.
GTP bound GDP bound eIF-2B
eIF2 switches from active form (___________) to inactive form (___________) through the association with _______
organelle
each complementary set of SNAREs is associated with a particular __________ involved in the secretory pathway
In prokaryotes, functionally related genes are usually clustered in a single operon. What has been found to be the case in eukaryotes?
each gene in a cluster of functionally related genes has its own promoter and is individually transcribed. Can be on the same chromosome or on different chromosomes.
EF-1 EF-2
elongation factors in eukaryotes; enter and leave the ribosome during each cycle
GTP hydrolysis
elongation factors use ___ _________ to displace the spent tRNA and reset the ribosome
Mutant bicoid (anterior) gene in drosophila mother=
embryo with posterior structures at both ends and no front half.
regulation of gene expression
enables cells to respond to environmental conditions
amelogenin
enamel matrix protein; coding gene is composed of 7 exons
lac Y
encodes a cell membrane protein called lactose permease to transport lactose across the cell wall
lac A
encodes a thiogalactoside transacetylase to get rid of the toxic thiogalactosides
elongation factors
enhance translation by: -driving it forward -controlling the accuracy
(eukTR) specific transcription factors bind to
enhancers
distal control elements
enhancers, maybe far away from a gene or even in an intron
Positive regulation
environmental conditions in the cell causes an activator protein to bind to the promoter site for a gene (or operon), which enables RNA Polymerase to begin transcription.
How can proteins be degraded and example (with nucleases):
enzymatic shortening of Poly-A tail & removal of 5' cap = nucleases chew up the mRNA. ex: cyclins that regulate the cell cycle need to be short lived for the cell to function properly. Marked by ubiquitin for destruction, this is recognized by proteasomes that degrade them.
the presence of the protein or RNA
essential for the function of the cell
General transcription factors (GTF) are _____ for all protein coding sequences. what do they do?
essential. They bind to other proteins and only when the whole transcription initiation complex is complete will RNA pol II begin.
attenuation
essentially the premature termination of an RNA molecule in the process of being transcribed
promoter
every gene has a __________, which is a binding site for the transcriptional apparatus, RNA pol, etc.
How can proteins be processed and example:
ex: cleavage of protein insulin = active insulin. Chemical modifications for functionality. Proteins for cell surface have sugars added to them
What happens when double-stranded RNA molecules are injected into a cell?
expression of a gene with the same sequence as the injected RNA is turned off. This is called RNA interference (RNAi) due to siRNAs.
diseases
failure in protein folding causes several known _______ such as CF and Sickle cell anemia
external signals
finely tuned genes can change in response to _____ _______ i.e. starvation leads to synthesis specialized enzymes in the liver->amino acids to glucose
(EukTR) general transcription factors are essential
for the transcription of all protein-coding genes -a few bind to the TATA box within the promoter -many bind to proteins, including transcription factors and RNA polymerase II
How are miRNAs formed?
formed from RNA precursors that fold back on themselves, forming short hairpin structures held together by Hbonds.
How are siRNAs formed?
from much longer double stranded RNAs, which give rise to many siRNAs.
genetic switch
gene transcription can be turned on and off in response to a variety of signals; this is termed:
repressible enzymes
generally function in anabolic pathways; synthesize essential end products from raw materials (precursors, enzymes)
E. coli prefers ________ over _______
glucose over lactose
stability
hairpin structure of miRNA provides _________
rough ER (RER)
has ribosomes on the cytosolic surface
139 nucleotides long
how many nucleotides is present in the leader transcript ? From start - Attenuator
decreased
if the distance between position two and position three were increased in the attenuator sequence of the trp operon then transcription would be ________ (increased/decreased)
increased
if the distance between the start of the leader peptide and sequence two of the attenuator were increased then transcription would be ________ (increased/decreased)
decreased
if the tryptohopan codons in the leader sequence of the trp attenuator were replace with histidine codons than transcription would be ________ (increased/decreased)
antiterminator
if tryptophan is low then the ribosome will proceed slowly along the mRNA strand of the leader sequence in the trp operon, which allows for formation of the ________
transcription factors
important DNA binding proteins that contain structural motifs that can "read" DNA sequences
eukaryotic initiation factors (eIF's)
important to initiate and thus control the the translation of mRNA
operator
in bacterial DNA, a sequence of nucelotides near the start of an operon to which an active repressor can attach; binding of the repressor prevents RNA polymerase from attaching to the promoter and transcribing the genes of the operon
cytoplasm
in eukaryotes translation occurs in the:
nucleus
in eukaryotes, transcription occurs in the:
Basal transcription factors definition (same as general)
in response to injections from activators, these factors position RNA polymerase at the start of transcription and initiate the transcription process
regulation of a metabolic pathway
in the pathway for tryptophan synthesis, an abundance of tryptophan can both inhibit the activity of the first enzyme in the pathway (feedback inhibition), rapid response, and repress expression of the genes encoding all subunits of the enzymes in the pathway, a longer-term response
lariat
in the second step of RNA splicing, the cut 5' end of the intro links to the adenine, creating a loop structure (________)
P site
in the second step of protein synthesis, a new peptide bond is formed at:
-OH group
in the third step of RNA splicing, a free ___ ______ then reacts with the start of the next exon, joining the 2 exons
E site
in the third step of protein synthesis, the mRNA moves 3 nucleotide positions through the ribosomal subunit to the:
the trpR gene is not located
in the trp operon
a molecule called an inducer
inactivates the repressor and turns of transcription (part of inducible operon)
the presence of lactose __________ the repressor, and turns the lac operon ______
inactivates, on
the trpR protein by itself is ______ and won't bind to _____
inactive, the operator
exon 4
inclusion of ________ ___ of the amelogenin gene is deleterious to enamel matrix formation
Can an individual TF work on more then one gene?
individual TF can act on several genes
chemical modifications to histones and DNA of chromatin
influence both chromatin structure and gene expression
epigenetic inheritance
inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence of a genome
in eukaryotic transcriptional regulation, repressors
inhibit transcription
repressors are proteins that
inhibit transcription
DNA methylation
inhibits transcription
initiation complex
initiation requires the ribosome to binds to the mRNA, which forms an _________ ________ containing the 1st aa-tRNA
AUG
initiation/start codon (____) sets the reading frame for protein translation
precursor protein
insulin is synthesized as a _______ _________ which contains the alpha and beta chain of insulin joined in a continuous single chain with an intervening C-chain
(eukTR) DNA is bent, which allows the transcription factors bound to enhancers to
interact with the transcription initiation complex and RNA polymerase
The choice between termination and anti-termination is often mediated by what?
interaction of a regulatory molecule with the RNA
The numbers of activator vs repressor TFs depends on?
intercellular signal control over their synthesis
translational ER
involved with budding that sends vesicles to the golgi
Operons
is a coordinated set of genes regulated as a single unit
Gene Expression
is tightly regulated, or controlled, so that the *cell only makes the gene products that it needs for efficient growth* under its current environmental conditions
The inactive mammalian X chromosome is heavily methylated. What is the result of this methylation?
it becomes inactive, and will stay this way not because it can't go back, but because the cell monitors is to stay untranscribable. This way different alleles of the same genes won't be expressed at the same time.
eIF2
key factor that binds the initiator Met-tRNA
Lactose permease
lac Y codes for?
The lac operon (and others) is regulated in response to _______ through _______ What does expression of these genes require?
lac operon (and others) is regulated in response to the availability of other carbon sources through catabolite repression Expression requires a transcriptional activator called CAP (catabolite activator protein)
What kind of catabolite regulation is the lac operon under? Why? What happens to the cell if glucose and lactose are present in the media because of this?
lac operon is regulated in response to other carbon sources --> catabolite repression ensures that genes for lactose utilization aren't expressed if another better carbon source is present like glucose Leads to a diauxic growth curve when glucose + lactose is present - The cell will use the glucose until it is gone then switch gene expression for lactose utilization (lag-log-short lag-log-stationary-death)
What are mutations in the lacZ or lacY genes considered and why? Can they be complemented?
lacZ and lacY mutations are Lac- because they don't make the B-galactosidase or the permease - but these mutations can be complemented because they are trans-acting so if there is a gene for either of these elsewhere in the organism lactose can be utilized.
What does a lacp mutation result in and can it be complemented? Why/why not?
lacp mutation is cis-acting: if the site on the DNA at which RNA pol initiates transcription is mutated --> transcription can no longer occur for the structural genes even if there is a lacp somewhere else in the cell
euchromatin
lightly-packed region, transcriptionally active
ribosomes
link amino acids together in the order specified by the codons of mRNA molecules to form polypeptide chains
TF regulate gene expression by...?
linking to one or more distal enhancers, which modulate promoter activity
DNA methylation can cause
long-term inactivation of genes in cellular differentiation
Post-transcriptional control includes regulation of mRNA degradation. Explain how this affects translation:
mRNA are degraded in cytoplasm a few minutes after synthesis in bacteria (hours/days/weeks in euk), allowing patterns of protein synthesis to change quickly in response to stimuli.
deadenylation nuclease (DAN)
mRNA can be degraded by an enzyme called:
small
mRNA is associated with the _____ ribosomal subunit
5'->3'
mRNA is read/translated in the _______ direction
What happens to the mRNA structure of a ribosome at high temperatures and what does this allow? What does temperature control overall in making the protein?
mRNA structure melts -> the Shine Dalgarno will be available -> ribosome can bind and translation can occur Making the protein - controlling whether the transcript can be read
bacteria cells don't waste energy
making tryptophan (if available in environment) -no need to go through transcription and translation to produce enzymes necessary to make tryptophan
transcription
many genes are regulated primarily at the level of:
histone acetyl transferases
many of the TATA binding protein Associated Factors are that make up the general transcription factor TFIID are _______ _______ _______ (HATs)
What are master regulatory genes, example, and what do they do?
master regulatory genes make proteins that will commit the cell to their destiny. ex: myoD protein stimulates expression of itself so the cell will stay this way.
Explain the distribution of cytoplasmic determinants for how different sets of activators come to be present in two cells:
maternal substances in the egg that influence early development. They're unevenly distributed in the cytoplasm of the egg, so different cells end up with different amounts of molecules as the cells divide.
RNA induced silencing complex (RISC)
mature miRNA is loaded into a protein complex called _________________________, where it is directed to the target mRNA to exert control of gene expression
miRNA
may also be involved in translation repression
How do cancer-causing viruses work?
may donate an oncogene to the cell, disrupt a tumor-suppressor gene, or convert a proto-oncogene to an oncogene. Some viruses also produce proteins that inactivate p53 and other tumor-suppressor proteins, making the cell more prone to cancer.
large
met-tRNA can only bind without the ________ ribosomal subunit attached
chromodomains
methylation of DNA increases the stability of the DNA-histone association and thereby decreases transcription. However, it can also increase transcription because remodeling complex proteins that disturbed the DNA histone association have ____________ that bind methylated residues on histone tails
heterochromatin
methylation promotes ________ formation
decrease
methylation, a type of post transitional modification that occurs to histone proteins to alter the rate of DNA transcription causes a __________ (decrease/increase) in the amount of transcription by making the histone protein more hydrophobic
transcribed translated
miRNA genes are _________ but they are not ________ into protein i.e. they are non-coding RNAs
heart disease
miRNA is associated with changes that occur in _______ ________- hypertrophy, fibrosis, arrhythmias
one or more
miRNA molecules have perfect (or near perfect) complementary base pairing to _____ ____ ________ target mRNA transcripts
osteogenesis
miRNAs play an important role in:
Inducer
molecule that stimulates synthesis of protein by binding with repressor
product of the Bicoid gene=
morphogen that determines the anterior end of a fly.
degeneracy of the code
most amino acids are represented by more than one codon
What are three mechanisms for converting a proto-oncogene to an oncogene?
movement of DNA within the genome, amplification of a proto-oncogene, and point mutations in a control element of the proto-oncogene itself. All result from broken and incorrectly rejoined chromosomes.
eukaryotic genes generally have
multiple control elements
A given gene may have _________ enhancers that are associated with ________________.
multiple enhancers that are associated with that gene and no other.
PAX9
mutations in _______ results in partial or total anadontia
control elements can be located
near (proximal) the promoter or at a distance (distal) from the promoter
In e. coli regulating trp synthesis, is it positive or negative inhibition?
negative because the rate decreases as concentration of product increases.
an inducible operon is usually
off
Is the repressor active by its self?
no its inactive, it need a corepressor
What are proto-oncogenes:
normal versions of oncogenes. They encode for proteins that stimulate normal cell growth & division.
miR-375
normally, ________ regulated pancreatic alpha-cell (glucagon producing), and beta-cell (insulin producing) mass. A lack of it causes beta cells to die, while alpha cells increase in mass
ATP dependent
nucleosome proteins can be added/removed/exchanged by the _____ _________ chromatin remodeling complexes
-histone N-terminal tails -histone H1 molecules
nucleosomes are further packed together by:
64
number of possible codons
RNA processing
occurs as soon as the transcript is being made
nucleosome
octomeric histone protein core made up of 2 each of H2A, H2B, H3, H4 histone proteins
general transcription factors
one of a set of eukaryotic proteins that are typically required for the synthesis of mRNA
histone acetylation _________ chromatin structure and ________ transcription
opens up chromatin structure and promotes transcription
the "switch" is a segment of DNA called the
operator
the trpR protein combined with tryptophan is active and will bind to the ________, blocking ______
operator, blocking transcription
prokaryotic eukaryotic
operators and silencer are both regions of DNA that are bound by repressor proteins that interfere with RNA polymerase and the rate of transcription. operators are found in __________ organisms whereas silencers are found in ________ organisms
What is an operon and is LacI considered to be apart of the lac operon?
operon = all the genes that are transcribed into the same mRNA and are under the control of the same promoter -- lac operon does not include the repressor which is adjacent to the lac Z, Y, and A genes
protein
operons are beneficial because they reduce the amount of unnecessary ________ that is produced since they produce proteins that have related functions which contributed to accomplishing a common functional goal.
What is controlled by homeotic genes?
pattern formation in late embryo, larva, and adult. Regulatory genes that cause placement of structures.
P site
peptidyl-tRNA is bound in the:
kinases
phosphorylate
elongation
polypeptide chain is extended by the sequential addition of amino acids
reversible
post-translational modifications are:
intronic exonic
pre-mRNA contains both _______ and __________ sequences
consensus sequences
pre-mRNA has conserved motifs (________ ________) at the junctions of exons and introns
What is the presence of a HTH motif in a protein usually indicative of?
presence of a HTH motif in a protein is a way to help identify it as a DNA binding protein
precursor mRNA (pre-mRNA)
primary mRNA transcripts in the nucleus are termed:
the operator is usually positioned within the
promoter
activated CRP attaches to the _______________ and ________________
promoter of the lac operon and increases the rate of transcription
List the 3 components of an operon and explain the role of each one:
promoter: where RNA pol binds to begin transcription. Operator: on/off sequence in or after promoter Operon Genes: functionally related genes controlled by the same operator
histone acetylation
promotes transcription
translational apparatus
ribosomes are also known as the:
Repressor
protein coded by the regulatory gene: prevents binding of RNA polymerase
histone
protein molecule around which DNA is tightly coiled in chromatin
reversible
protein phosphorylation is a reversible PTM mediated by kinases and phosphatases
rRNA tRNA mRNA template
protein synthesis involved interactions between 3 types of RNA molecules:
Argonaute
protein that is part of RISC that binds to one strand of the miRNA molecule
Explain how proteins are degraded with proteasomes:
proteins are marked for destruction by ubiquitin molecules (requires ATP). Proteasome protein complexes then see this tag, unfold the protein, and put it in their central cavity. Enzymatic components of the proteasome then cut the protein into small peptides which can be further degraded by other enzymes in the cytosol.
constitutive secretion
proteins are secreted from a cell continuously, regardless of external factors or signals
regulated secretion
proteins are secreted from a cell when a specific signal is detected by the cell i.e. insulin secretion
specific transcription factors
proteins that bind to control elements associated with a particular gene and, once bound, either increase or decrease transcription of that gene
mediator proteins
proteins that mediate the interaction between regulatory proteins and the transcription factors
Tumor suppressor genes code for...
proteins that prevent uncontrolled cell growth. Some TS proteins (TSP) repair damaged DNA, or control adhesions of cells to each other or to the ecm, or are components of cell signaling pathways that inhibit the cell cycle. Any mutation that decreases the amount of TSP increases the chance of cancer.
irreversible
proteolysis is:
chromatin-modifying enzymes
provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machinery
nucleus cytoplasm
rRNAs, tRNAs, and mRNAs are transported from the ________ to the __________ where these molecules carry out their cellular functions
initiation
rather than blocking __________ of transcription, as is accomplished buy binding of trp repressor to the operator sequence, attenuation prevents completion of transcription.
ubiquitination
regulated PTM; regulated degradation mechanism to control protein levels
eukaryotic gene expression
regulated at multiple levels
transcription
regulation of gene expression in prokaryotes is primarily accomplished through regulation of ______
prokaryotes
regulation of genes expression in _________ is primarily accomplished through regulation of transcription
Repression
regulatory mechanism that inhibits gene expression and decreases the synthesis of enzymes
In addition to affecting mRNAs, siRNAs can cause......
remodeling of the chromatin structure. siRNAs form heterochromatin at centromeres. They bind to a certain protein complex and bring it to the centromere, where they recruit enzymes to make that area highly condensed heterchromatin.
Nucleosome
repeating subunit of chromatin fibers, consisting of DNA coiled around histones
histone acetylation
results in loose packing of nucleosomes; transcription factors can bind the DNA and genes are expressed
What does the rpoH gene encode? What happens to its transcript in low vs. high temperatures?
rpoH encodes the heat shock sigma factor sigmaH Low temp: inactive mRNA -> no translation High temp: active mRNA -> translation
4
sequence 3 is partially complementary to sequence_________
codon
sequence on mRNA is read in groups of 3 nucleotides:
tRNAs
serve as adaptors between the mRNA template and the amino acids being incorporated into protein
Repressible
several genes in a series are turned off by the product synthesized by the enzyme and contains coding for anabolic enzyme
disease biomarkers
several miRNAs have links with types of cancer; this potential for miRNAs to act as _______ __________ will aid in treatment
microRNAs (miRNAs)
short RNA molecules that can bind to 3' UTR of other mRNAs
binding of a repressor to the operator
shuts off transcription
Promoter
site in the DNA which the RNA polymerase recognizes as an initiation signal to indicate where transcription of RNA begins
Operator
site in the Dna which repressor binds
Operator
site in the Dna which repressor binds. When bound to repressor, transcription is prevented
Corepressor:
small molecule that cooperates with repressor protein to switch operon off
microRNA (miRNA)
small single-stranded regulatory RNA molecules that regulate gene expression
initiation
small subunit of mRNA binding site is joined by large subunit and aminoacyl-tRNA binds
secretory vesicles
small, membrane-enclosed transport vehicles, that transfer proteins assembled in the ER to the Golgi, and then to their appropriate cellular destination/location
aminoacyl-tRNA synthetase
specific __________ _______ enzymes link a specific tRNA with a specific amino acid
Histone code hypothesis:
specific combinations of modifications, rather than the overall level of histone acetylation, help determine the chromatin configuration, influencing transcription.
Lac operon
this is a negative (because absence of lactose) inducible transcriptional control system
Explain how the lac operon is under dual control:
stimulated by CAP, blocked by lac repressor. CAP controls rate, lac repressor controls if translation occurs at all.
What are the structural genes in the E.coli ara operon and what promoter are they controlled under? What mediates regulation and where?
structural genes include araB, araA, and araD which are transcribed from a single promoter pBAD upstream of the promoter is the activator region, araI, where the activator protein AraC binds to activate transcription in the presence of L-arabinose and the CAP site which CAP binds to mediate catabolic regulation
disulfide bridges
structural post-translational modification; linkage of a sulfur between 2 cysteine amino acids
What are morphogens?
substances that establish an embryo's axes and features.
The lactose operon is likely to be transcribed when
the cyclic amp and lactose levels are both high in the cell.
morphogenesis
the development of body shape and organization
What is meant by determination? Explain what this means within an embryonic cell:
the events that lead to the observable differentiation of the cell. Not reversible, even if the determined cell is placed elsewhere in the embryo. Cells have become noticeably different in structure and function. Cells express the genes they'll need in the tissue they're destined for (tissue specific proteins, ex: liver makes albumin).
what is differential gene expression?
the expression of different genes by cells with the same genome
differential gene expression
the expression of different sets of genes by cells with the same genome
promoter
the gene _________ contains DNA sequences that are capable of binding transcription iniating proteins
transcription terminator
the hairpin formed between positions three and four in the trp attenuator sequence within the leader sequence is referred to as a _________ ________
HOX genes determine
the identity of embryonic regions along the antero-posterior axis: i.e. body segmentation
operator
the lac operon has multiple ________ sequences. This is advantages because it allows for maximal repression of transcription to reduce unnecessary protein production.
operator structural
the leader sequence in the trp operon is located between the _______ and the ________ genes
eIF-4
the mRNA transcript associates with _________ proteins at the 5' cap and 3' polyA tail of mRNA; allows for correct positioning in small ribosomal subunit
RNA transcript
the new _____ _________ is released from the polymerase as it is synthesized. This allows the RNA to be processed as soon as it's made
Within a cell, the amount of protein made using a given mRNA molecule depends partly on
the number of introns present in the mRNA.
Inducible
the operon is turned in by the substrate of the enzyme for which the structural genes code (ex. lactose)
DNA helix
the outside of the _______ ________ can be read by TF's
transcription initiation complex
the part of a gene's promoter where synthesis of the gene's RNA transcript begins.
histone code
the pattern of histone modifications in termed the _____ ______ and has specific meanings
methylation
the phenomenon in which genes are silenced through ________ depending on which parent they are inherited from is called imprinting
Expressor proteins (Activator protein)
the positive transcriptional control system codes for?
(eukTR) for genes not expressed all the time, high levels of transcription depend on..
the presence of additional, specific transcription factors
induction
the process in which one group of embryonic cells influences the development of another, usually by causing changes in gene expression
gene regulation
the process of controlling which genes in a cell's DNA are expressed
determination
the progressive restriction of developmental potential in which the possible fate of each cell becomes more limited as an embryo develops
operators are sequences of nucleotides between
the promoter and the transcriptional start where active repressors can bind
chromodomain
the region of the remodeling complex that binds the methylated chromatin is referred to as the _________
operator
the regulatory sequence of the DNA downstream of the promoter, which can bind repressor proteins that prevent the transcription of the structural genes by RNAP, is referred to as an _________
histone deacetylation
the removal of acetyl groups to certain amino acids of histone proteins
A site P site E site
the ribosome has 3 tRNA binding sites:
acetyl coa
the source for histone acetylation is ________ _______
proofreading reactions
the specificity of amino acid attachment to tRNA is controlled by _______ ________ that will hydrolyze any incorrectly formed aa-tRNAs
cell differentiation
the structural and functional divergence of cells as they become specialized during a multicellular organism's development; depends on the control of gene expression
How do transcription factors work? (General)
they bind to the promoter regions of DNA or directly to RNA polymerase to begin transcription.
cells stop the digestion of lactose b/c
they prefer to digest glucose
Excessive nutrient
this acts as a co repressor that is needed to block the action of an operon
Lactose
this acts as an inducer to turn on the transcription of a gene for the production of the enzymes necessary for utilization
E. coli
this bacteria can use either glucose or lactose for environemntal adaptation
Operon
this controls an important biochemical process and is only found in prokaryotes
cAMP Receptor Protein (CRP) and Catabolite Activator Protein (CAP)
this controls the attachment of RNA to the promoter
Thiogalactoside transacetylase
this gets rid of the toxic thiogalactosides
Transcriptional Regulation
this happens by controlling the access of the RNA polymerase to the promoter
Diauxic Growth
this happens when the glucose rans out (What do you call the lag phase)
Lactose
this induces the expression of the lac genes
Explain inducible operons and describe one example
transcription is sually off but can be induced when a small molecule binds to regulatory protein. Inducer inactivates the repressor. Ex: the lac operon
Explain repressible operons and describe one example
transcription is usually on but can be inhibited when small molecule binds to regulatory protein. Repressor molecule activates the repressor protein. Ex: the trp operon
repressible operon
transcription is usually on, but can be inhibited (repressed) when a specific small molecule binds allosterically to a regulatory protein (example tryptophan)
repressor
transcription of the tryptophan synthetic enzymes in the trp operon is regulated by the trp ________, which binds tryptophan to form a complex that specifically binds the operator.
operator
transcription of the tryptophan synthetic enzymes in the trp operon is regulated by the trp repressor, which binds tryptophan to form a complex that specifically binds the _______.
The functioning of enhancers is an example of
transcriptional control of gene expression
How do transcriptional regulators usually bind DNA? What is this structure composed of and how does this structure allow for DNA binding?
transcriptional regulators usually bind to DNA often through their helix turn helix motifs (HTH) - Two regions of approximately 7-9 amino acids that form an alpha-helical structure which is helix 1 and 2 - helix 1 and 2 are separated by 4 amino acids - When the protein binds to the DNA, helix 2 lies in the major groove of the double helix so the amino acids in helix 2 are able to make contact with specific bases in the DNA and form hydrogen bonds
exosomes
transfer molecules from one cell to another via membrane vesicle trafficking
termination
translation ends by stopping the addition of amino acids. the completed protein is released and the ribosome dissociated from the mRNA
translation repressor proteins
translation initiation can be blocked by _____ ____ _______ which hide the Kozak sequence
ribosomes
translation is carried out on:
the trpR protein combined with ___________ is ________
tryptophan, active
1. DNA methylation 2. Histone Modifications
two main types of epigenetic modifications
in euk transcriptional regulation, transcription factors
typically have a DNA binding domain and a protein interaction domain
proteasome
ubiquitination marks proteins for degradation via the:
genes with highly packed heterochromatin
usually not expressed
miRNA biosynthesis pathway
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Attempts to stop the ribosomes attempting to translate the leader peptide. The ribosome stops at the trp codons when [Tryptophan] is too low. The stopped ribosome prevents a downstream transcription of terminator from forming.
what is the function of tryptophan codons in the leader peptide?
negative control
when TF's bind to DNA and turn genes off
positive control
when TFs bind to DNA and turn genes on
coordinate control:
when a single on/off switch can control a group of genes with the same function (transcription unit)
the lac operon is off
when lactose is absent
the lac operon is on
when lactose is present
all organisms must regulate
which genes are expressed at any given time
compounds present in any given cell determine
which genes are switched on
Because the 1 is already formed certain hairpin with 2. So 2 is not present to hybridize with 3 (Same goes with other hair pins)
why is the hybridization of 1 and 2 to form the 1-2 structure prevents the formation of the 2-3 structure?
