Regulation Of Gene Expression Flashcards
What is constitutive gene expression?
Gene products that stay more or less constant
Also called house keeping genes
What are the two levels of control for gene expression?
Transcription and translation
What are the 3 ways to measure gene expression?
Northern blot
RT-PCR
DNA microarray
How is RT-PCR done?
- Blood sample from infected person (i.e. HIV)
- Remove cells by centrifugation
- extract viral RNA genome
- Use reverse transcriptase/ PCR amplification
- Do Gel electrophoresis with a control (blood from non infected person)
What is the major determinant for expression of house keeping genes?
Differences in promotor sequences can account for 1000-fold difference in mRNA product
What does deviation from consensus sequence usually do to promotor function?
Reduce expression
What do specifity factors do? Examples?
Alter specificity of RNA polymerase for promotors
I.e.
Sigma factors in bacteria
TBP protein in eukaryotes
What are repressor regulatory proteins? What kind of regulation is it?
They impeded access of RNA polymerase to the promotor.
This is negative regulation
What are activator regulatory proteins? What kind of regulation?
These enhance RNA polymerase-promoter interaction.
This is positive regulation
What are the 3 classes of regulatory proteins?
- Specificity factors
- Repressors
- Enhancers
What sigma factor recognizes most promotor genes?
Sigma factor 70
What sigma factor recognizes genes induced by heat shock?
Sigma factor 32
What sigma factor recognizes gene for stationary phase and stress response?
Sigma factor 28
Do the same sigma factor recognizse the -10 and -35 region on bacteria?
No
What sigma factor recognizes genes involved in motility and chemotaxis??
Sigma factor 28
What sigma factor recognizes gens for nitrogen metabolism?
Sigma factor 54
What sigma factor recognizes genes dealing with misfolded proteins in the periplasm?
Sigma factor 24
What are the things that can act on a repressor protein?
Co-repressor : enhances repressor
Inducer: reduces binding of repressor
What does a co-repressor do?
It enhances the binding of the repressor to the operator
What does an inducer do?
It reduces the binding of the repressor to the operator
What are the molecules that can act on activator proteins?
Co-activator: enhances binding of activator
Repressor: reduces binding
What is a co-activator?
When the signal molecule enhances the binding of the activator to the promoter
What is a repressor in respect to an activator?
When the signal molecule reduces the binding of the activator to the promoter
What is an operan? (Only in prokaryotes)
The promoter region which contains binding site for RNA polymerase (sigma subunit) and adjacent binding sites for activators and repressors.
- The repressor binding sequence is the “operator”
- (cluster of genes involved in the same biochemical pathway)
What produces polycistronic mRNA’s?
Operons
What does the tryptophan operan consist of?
The promoter region and 5 genes involved in tryptophan biosynthesis
What does the promoter of tryptophan have? What kind of promoter is it?
A 15-pair region recognized by a protein.
This protein inhibits further expression and is therefore a repressor
The repressor for the tryptophan operan requires what to bind efficiently to the operator sequence?
Two Trp’s
Trp acts as a co-repressor (it enhances the binding of the repressor)
The repressor is always present in ~20 copies per cell via what kind of expression?
Constitutive expression
Note: this is the house keeping kind of expression
What molecules affect the lac operon?
Glucose and lactose
What is the preferential carbon source for E. coli?
Glucose
What will E. coli use in the absence of glucose?
Lactose
In E. coli the use of lactose coincides with the expression of what gene?
Beta-galactosidase expression
What occurs when both glucose and lactose are present in respect to lac operon?
Operon off because CAP not bound
What occurs when there is glucose (+) and no lactose (-) in respect to the Lac operon?
Operon off
Because Lac repressor bound and because CAP not bound
What happens when both glucose (-) and lactose (-) are not present in respect to the Lac operon?
Operon off because Lac operon is bound
What happens when glucose (-) is not present but Lactose (+) is present in respect to the Lac operon?
Operon on
What does lacZ code for?
Beta-galactosidase
What does LacY code for?
The lactose permease
What does LacI code for?
A constitutively expressed repressor
What does allolactose act as?
An inducer
Binding of allolactose to the repressor results in?
An inactive repressor that has no affinity for the operator
What is cAMP in respect to CAP?
A co-activator
The Lactose operon can only work when it has been first properly activated by?
The cAMP Responsive Protein (CRP) aka the catabolic catabolite activator protein (CAP)
(It must bind cAMP to function)
What kind of effect does glucose have in respect to cAMP?
It lowers intracellular cAMP levels by inhibiting adenylyl cyclase, causing CAP to loose its bound cAMP
-This is called the “glucose effect” or “catabolite repression”
Attenuation is a mode of transcriptional regulation of operons for ____________.
Amino acid biosynthesis
What is transcription attenuation?
Inhibition of gene expression by premature termination of transcription
Upstream of the structural genes in the operon lies a short “open reading frame” (ORF) of about 42 base pairs that functions as?
Codons for particular amino acid whose biosynthesis is directed by the operon
In prokaryotes when does translation start? (In respect to transcription)
Before transcription is completed
The Trp repressor can reduce expression by a factor of?
70
Attenuation can decrease expression by a factor of?
10
When cellular tryptophan concentration is sufficient, the transcription of the trp operon is repressed by?
700 fold
70 from Trp repressor * 10 fold from attenuation = 700
The nucleotide sequence of the 5’ -end of trp mRNA includes the short ORF that encodes a 14 amino acid peptide containing two Trp residues and includes an untranslated attenuator region that can engage in what?
A hair pin formation
Note: The 5’ end is called the Leader region of trp mRNA
What kind of hairpin structure is favored in the absence of obstructions?
2:3 pair
Note: the other hairpin is the 3:4 (atternuator)
What doe high tryptophan levels promote?
The 3:4 hairpin
When there is a 3:4 hairpin from high tryptophan levels what occurs?
Enables Rho-factor independent termination of transcription
When tryptophan levels are high, the ribosome _____ translates sequence 1 (open reading frame encoding leader peptide) and blocks sequence 2 before sequence 3 is transcribed.
Quickly
Note: continued transcription leads to attenuation at the terminator-like attenuator structure formed by sequence 3 and 4.
Activator binding sites are usually _______ of the RNA polymerase binding site.
Upstream
The tryptophan operon is under _____ regulation control in which the molecular signal ( Trp) causes binding of the ______ to the operator.
Negative
Repressor
The lactose operon is under _____ control by lactose and under ____ control for glucose.
Negative
Positive
What causes dissociation of the repressor from the Lac operon?
Lactose (through allolactose)
Glucose (through cAMP) affects binding of ____ (aka ____) to the activator binding site.
Note: high glucose: low cAMP (and vice versa)
CRP (aka CAP)
Regulation of gene transcription is mediated by _____ and/or ______ in concert with molecular signals.
Repressors and/or activators
_________ inhibit operon transcription by binding to a site (operator site) close to the binding site of the RNA polymerase.
Repressors
Attenuation works an an _____ inhibiting transcription that has escaped repressor mediated inhibition
Extra brake
The _______ for genes encoding enzymes involved in amino acid biosynthesis consists of the ORF for the leader peptide and three regions which can engage in a proximal or distal hairpin (downstream of the ORF coding for the leader peptide.
Attenuation region
When there ______ of the amino acid the operon codes for, then the leader peptide is readily synthesized and the distal hairpin is favored, resulting in premature termination of the transcript
Is enough
When there is ________ of the amino acid the operon codes for, the ribosome stall at the sequence coding for the leader peptide (the codons waiting for charged-tRNA)- resulting in complete transcription of the operon
Not enough
DNase hypersensitivity sites differ from tissue to tissue for selected genes: this reflects?
Cell type-specific gene expression
What are the two clases of enzymes that play a role in chromatin remodeling?
- Enzymes that covalently modify the core histones of nucleosomes : Histone Acetyl Transferases (HATs) and Histone DeAcetlyases (HDACs)
- Enzymes that use ATP to remodel nucleosomes on the DNA: SWI/SNF that reposition or remove nucleosomes
What do SWI/SNF complexes do?
Reposition or remove nucleosomes
-create nucleosome-free regions in the chromosome and thus allow for binding of transcription factors
Acetylation status of amino-terminal domains of histone H3 and H4 determines the affinity between histone core particles and DNA. What is responsible for these modifications?
Histone Acetyl transferase (HATs)
Histone Deacetylases (HDACs)
What mutation is involved in Rubinstein-Taybi syndrome?
Caused by mutations in the CBP protein or in the closely related p300 protein both of whom have HAT activity
What are the characteristics of rubinstein-Taybi syndrome?
Characterized by postnatal growth deficiency, microcephaly, specific facial characteristic, broad thumbs and big toes and mental retardation
What kind of mutation is Alpha-thalassemia/mental retardation syndrome?
Caused by mutations in the ATRX protein that is a member of the SWI/SNF family
What are the characteristics of alpha-thalassemia/mental retardation syndrome?
Patients are mentally retarded, have characteristic facial appearance and have a mild form of alpha-thalassemia while their globin genes are normal
Enhancers may be thousand of base-pairs away from the transcription initiation site and make contact with the initiator sites though __________.
Transactivation
______ allows for interactions between distant DNA sites (HMG proteins)
DNA looping
What are cis acting regulators?
Regulatory elements like promotors and enhances that act on the same chromosome as the transcribed gene
What are trans acting regulators?
Activator/repressor proteins that diffuse to the action