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
25
CTD requirement
- Coordination of transcription and processing by phosphorylating C terminal domain of RNAP II. Phosphorylation recruits capping enzyme (guanyl transferase and SAM) at end of CTD tail, splicing factors at middle, and polyadenylation factors (CPSF and CF) at beginning. Reflect sequence of capping, splicing, then polyadenylation.
