Control of Gene Expression Flashcards
1
Q
Repressor and Activators
A
- Repressors inhibit transcription
- Activators promote transcription
- Regulatory proteins are allosteric: possess sites that can be bound by both, separate from active site
- Can enhance or diminish activity of activator or repressor
2
Q
Positive Transcriptional Regulation
A
- When activator binds, facilitate recruitment of RNA polymerase and enhance activity of downstream gene
- Ligand can inactivate activator and dissociate from DNA
- Other activators require ligand to activate transcription
3
Q
Negative transcriptional regulation
A
- Repressor binds promoter -> inhibit RNAP binding
- Absence of repressor binding can transcription occur
- Presence of ligand can dissociate repressor and initiate transcription
- Other repressors need ligand to inhibit transcription
4
Q
Lac operon in prokaryotes
A
- Operon is functional unit of genomic DNA that consist of cluster of genes under single pronoter
- Lac operon is for metabolization of lactose to glucose
5
Q
Lac operon structure
A
- Transcriptional control region: Contains promoter for RNAP binding and operator for repressor binding
- Three related structural genes: lac z, lac y, lac a
- Upstream of lac operon is regulatory gene with lac i repressor -> cannot metabolize lactose -> lac i binds to operator region and inhibit expression
- Lac z encode beta-galactoside, Lac y encode transport protein galactosidase permease (brings lactose into cell), lac a encodes galactosidase transacetylase (acetylates any unhydrolyzed lactose for elimination)
- Direct relationship between lac operon and cell growth
6
Q
Negative regulation of lac operon
A
- Lac i repressor binds as dimer to operator region preventing RNAP from transcribing lac z, y, a
- One dimerized repressor binds to upstream promoter and another to downstream
- Forms tetrameric protein structure: DNA loop
- Repressors recognition helix contains arginine residues that transiently H-bond with base pairs of DNA
7
Q
Ligand binds repressor
A
- When lactose present, lac i dissociates
- Allolactose binds repressor, induces disorganization of repressor recognition helix, reducing affinity -> repressor dissociates
- Allolactose made from side reaction of lactose, beta-galactosidase changes glycosidic link between lactose monomers from 1-4 to 1-6
- High lactose in cell, high allolactose
8
Q
Positive regulation of lac operon
A
- When glucose absent, cells increase expression of lac operon using catabolite active repressor (CAP)
- CAP is dimeric protein, when activated, bind to DNA via C terminus, and N terminus bind to ligand cAMP, active when binded to two molecules of cAMP
- CAP bind upstream of lac promoter and assist in forming closed promoter complex to initiate transcription
- Amount of cAMP and glucose in cell inversely related
9
Q
DNA binding proteins in eukaryotes
A
- DNA binding proteins recognize specific DNA sequences
- 80% of DNA binding proteins characterized with helix turn helix, zinc finger, leucine zipper
10
Q
Helix turn helix
A
- Composed of several alpha helices
- Recognition helix that interacts with major groove of DNA
11
Q
Zinc finger
A
- Consist of zinc ion coordinated to two conserved histidines and cysteines
- Allow DNA binding protein to fold into compact structure, allowing for zinc finger alpha helix to interact with major groove
- Amino acids in helix bind to bases via H-bonds
12
Q
Leucine zipper
A
- DNA binding regions: Consist of basic residues of lysine and arginine, which are positively charged. Allow to interact with negatively charge backbone of DNA
- Connector region: 6 amino acid connector that hold DNA binding and zipper regions together
- Leucine zipper: Coiled structure formed by hydrophobic interactions between two alpha helices. Driven by hydrophobic leucine residues in both helices once every 7 amino acids
13
Q
Structure of estrogen and estrogen receptor
A
- Estrogen is hormone derived from cholesterol, acts as ligand to estrogen receptor
- Estrogen receptor is soluble and found in nucleus of cell, undergoes conformational change upon ligand binding, subsequently able to bind coactivator
14
Q
Estrogen receptor structure
A
- Transcription activation region: Helix 12 folds onto side of receptor and co activator able to bind to transcriptor activation region
- DNA binding region: Two zinc fingers bind to consensus sequence called estrogen receptor elements. bind as a dimer
- Hormone binding pocket: Hormone binding regions forms pocket that estrogen can bind to
15
Q
Transcriptional activation via estrogen receptor
A
- Receptor can bind DNA in presence or absence of ligand
- Estrogen binding induces conformational change in ER
- Co activator facilitates remodeling of chromatin bound by ER, allowing for RNAP binding, enhancing transcription of ER specific genes
16
Q
Drugs targeting hormone receptors
A
- Estradiol is type of estrogen hormone that acts as agonist of estrogen receptor -> receptor signaling pathway initiated
- Tamoxifen is antagonist, has hydrophobic rings that allow it to fit into ER binding pocket. Blocks H12 from folding, blocking co activator recruitment -> inhibit gene expression
- Tamoxifen used as breast cancer treatment
17
Q
rRNA processing
A
- RNA Polymerase 1 synthesizes single precursor rRNA including 18S, 5.8S, and 28S rRNAs
- 5.8S and 28S incorporated into large ribosomal subunit, and 18S in smaller subunit
- Nucleotide modifications made by small nucleolar ribonucleoproteins (snoRNPs)
- snoRNP complexes composed of rRNA and proteins modify base by adding methyl groups and converting uracils to psuedouracils
- Non modified spacer regions cleaved to produce the three mature rRNA molecules
18
Q
tRNA
A
- Synthesized by RNA Polymerase III
- RNAse P cleaves leader region at 5’ end
- RNAse D cleave trailer region at 3’ end
- Amino acid attachment site added at 3’ end by tRNA nucleotidyl transferase
- Base modification made: methylation of D loop and formation of psuedouridine
- Intron near anticodon loop spliced to generate anticodon
19
Q
mRNA
A
- Synthesized by RNA Polymerase II
- Eukaryotic mRNA exclusively monocistronic, each mRNA molecule only encodes one polypeptide product
- Prokaryote mRNA is polycistronic
20
Q
Capping
A
- 7-methyl G cap at 5’ end consist of guanine methylated at position 7. m7G not part of original transcript, connected by unique 5’-5’ triphosphate bond
- Several bases at 5’ end are methylated at 2’-OH position, are part of mRNA sequence
- Capping process begins while RNA polymerase still transcribing mRNA
- Added cap increase stability and protect mRNA
- Guanyl transferases: Guanyl transferase hydrolyzes gamma phosphate from 5’ end of transcript to release phosphate. Oxygen of 5’ beta phosphate attacks alpha phosphate of GTP causing release of pyrophosphate. Creates 5’-5’ triphosphate bond. Not yet methylated
- S-adenosyl methionine: SAM is cosubstrate that serve as source of methyl group for cap. Cap 0 methylated at position 7 of purine ring. Downstream nucleotides methylated at 2’OH position of ribose sugars
21
Q
Poly(A) tail
A
- Added at 3’ end through polyadenylation
- RNA polymerase stalling: Stalls at invariant U/G site located 10-35 nucleotides beyond poly A addition sequence(AAUAAA). Prompts recruitment of Cleavage and Polyadenylation Specificity Factor (CPSF) to mRNA
- Looping of transcript: CPSF binds to consensus sequence and invariant G/U causing looping
- mRNA cleavage: CPSF recruit cleavage factors CFs to cleave looped portion, transcript synthesized past Poly(A) is released
- Addition of poly(A) tail: CF dissocate after cleavage, CPSF recruit poly(A) adenylation protein to 3’ end. PAP adds 80-250 non template adenine
- Release of mature mRNA transcript: CPSF dissociates and leaves transcript with both 5’ and 3’ modifications
22
Q
RNA editing
A
- Specific nucleotides can be modified
- Increase protein diversity by altering amino acid
- Apolipoprotein (fatty acid and steroid transport protein): in liver, remain unedited to translate full lenght ApoB-100 protein with lipoprotein binding domain and LDL receptor
- If in small intestine, specific cytosine deaminated to uracil, introduces premature stop codon, producing ApoB-48 without LDL receptor domain
23
Q
Splicing
A
- Alternative splicing allows mRNA to be spliced in ways including or excluding different exons. Result in 2n isoforms, where n is number of exons
- Constitutive splicing removes introns and ligates exons. Require invariant 5’ GU splice site, key A residue branch site, pyrimidine tract near 3’ end, invariant 3’ AG splice site
- Occurs in two transesterification reactions. Reactions break phospho-ester bond between nucleotides and reforms new bond with different nucleotides
- Rely on small nuclear ribonuclear proteins (snRNPs), consist of snRNA and proteins: key U1, U2, U4, U5, U6 form spliceosome
24
Q
Mechanism
A
- Recognition of 5’ splice site: U1 snRNP contain specific snRNA with six conserved nucleotides -> base pair with 5’ splice site. Only ATP independent step
- Recognition of branch site: U2 binds to branch site, require one molecule of ATP for hydrolysis
- Complex binding: U4, U5, U6 complex replaces U1, U1 dissociates. One molecule of ATP used to make complex, second ATP used to facilitate binding
- Dissociation of U4: Once U5 is aligned at 5’ splice site, U4 dissociates. U2 + U6 catalytic site form across intro via base pairing interactions. U4 act as inhibitor, masks activity of U6 and prevent formation of catalytic site. One molecule of ATP consumed
- First transesterification reaction: U5 use one ATP to align 2’OH of A branch site to 5’ splice site. Breaks phosphodiester bond at 5’ site resulting in lariat intermediate. Adenine nucleotide of lariat intermediate has 5’-3’, 3’-5’, 2’-5’ phosphodiester linkages
- Second TE reaction: U5 align 3’OH of exon 1 to 3’ splice site using ATP. Reaction break phosphodiester bond between end of intron and beginning of exon 2, form bond between exon 1 and 2. Form lariat intron and splice product
- Release of lariat: U5, U6, and U2 and lariat require single molecule of ATP to dissociate. Final ATP required for dissociation at catalytic center.
- After splicing complete, ATP dependent helicase unwind RNA duplex formed by U6 and U2 facilitating dissociation
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