Exam 2 Review Flashcards
(165 cards)
1
Q
Describe how to identify membrane-spanning protein in genomic sequence
A
Membrane spanning proteins need to have regions of hydrophobic amino acids
Hydropathy plots can identify membrane spanning domains in proteins
Blocks of 20 amino acids are examined and the ratio of hydrophobic to hydrophilic amino acids calculated
2
Q
Gene knock-out
A
Disrupts the function of a specific gene by deleting or inactivating it.
Achieved through techniques like homologous recombination, CRISPR-Cas9, or RNA interference.
Allows studying the effects of gene loss on phenotype and elucidating gene function.
3
Q
Gene knock-in
A
Introduces a specific genetic modification into a target gene.
Typically involves inserting a transgene or modifying an endogenous gene sequence.
Used to study gene function, model genetic diseases, or engineer organisms with desired traits.
4
Q
Meganucleases
A
also known as homing endonucleases
An approach to specific cleavage
Contain both the DNA recognition and cleavage functionalities
Large recognition site (dsDNA sequences of 12-40 bp)
In nature they are expressed in archaebacteria, bacteria, phages, fungi, yeast, algae, and some plants
5
Q
Zinc finger motif
A
a common DNA binding domain
Can recognize about three base pairs specifically
The human genome contains about 700 zinc finger proteins, almost all of these are transcription factors
To make a protein with higher sequence specificity: fuse several zinc finger domains together
6
Q
Zinc finger nucleases (ZFNs)
A
Engineered proteins used for targeted genome editing.
Comprise zinc finger DNA-binding domains fused to a DNA-cleavage domain (e.g., FokI endonuclease).
Zinc finger domains recognize specific DNA sequences, guiding the nuclease domain to cleave at desired genomic sites.
7
Q
Structure of zinc finger domains
A
Consist of zinc ions coordinated by cysteine and histidine residues.
Each zinc finger recognizes and binds to 3-4 DNA nucleotides.
Multiple zinc fingers can be combined to create a modular DNA-binding protein with extended specificity.
8
Q
Controlling sequence-specificity (zinc finger)
A
Designing custom zinc finger arrays with desired DNA-binding specificities.
Utilizing bioinformatics tools to predict zinc finger-DNA interactions and optimize target site selection.
Incorporating modifications such as obligate heterodimerization or obligate homodimerization to enhance specificity and reduce off-target effects.
9
Q
Transcription activator-like effectors (TALEs)
A
Proteins derived from plant pathogenic bacteria, capable of binding to specific DNA sequences.
Composed of repeating modular units, each recognizing one DNA base pair.
Used for targeted genome editing, gene regulation, and protein localization.
10
Q
TALENs (TALE nucleases)
A
Fusion proteins combining TALE DNA-binding domains with a DNA-cleaving domain (e.g., FokI endonuclease).
Enable precise genome editing by inducing double-strand breaks at specific genomic loci.
Used for targeted gene knockout, gene correction, and site-directed mutagenesis in various organisms.
11
Q
Features and Uses of TALEs and TALENs
A
Highly specific and programmable, allowing precise targeting of desired DNA sequences.
Enable efficient and customizable genome engineering without requiring extensive protein engineering.
Widely used in research, biotechnology, and therapeutic applications for precise manipulation of genetic material.
12
Q
Describe how bacteria use CRISPR/Cas-9 to fight viruses
A
Bacteria incorporate viral DNA sequences (spacers) into their own genome within CRISPR loci.
CRISPR loci are transcribed and processed into CRISPR RNAs (crRNAs).
crRNAs guide Cas9 nucleases to complementary sequences in invading viral DNA.
Cas9 cleaves and degrades viral DNA, preventing viral replication and defending against viral infection.
13
Q
Describe how to use CRISPR/Cas-9 to cut any DNA sequence
A
Design guide RNA (gRNA) complementary to target DNA sequence.
Form ribonucleoprotein complex by combining gRNA with Cas9 protein.
Deliver CRISPR/Cas9 complex into target cells using methods like transfection or viral vectors
Cas9 binds to target DNA sequence guided by gRNA, inducing double-strand breaks (DSBs).
DSBs trigger cellular DNA repair mechanisms, such as non-homologous end joining (NHEJ) or homology-directed repair (HDR).
NHEJ leads to random DNA insertions/deletions, disrupting target gene function.
HDR allows precise DNA editing by incorporating exogenous DNA template during repair process.
14
Q
other uses of CRISPR/Cas-9
A
multiplexing, gene editing, genome editing
15
Q
CRISPR/Cas-9 advantages over other systems
A
DNA recognition depend on sgRNA instead of protein domains
Very easy to design/clone sgRNAs
Highly specific
Homozygous targeting
Multplexing
16
Q
multiplexing
A
Gene targeting with ZFN and TALENs require multiple rounds of editing for multiple genes
CRISPR/Cas9 can be multiplexed-simply adding multiple sgRNAs
17
Q
CRISPR/Cas9 applications
A
Gene expression repression (inhibition) = CRISPRi
Gene expression activation = CRISPRa
Painting the genome CRISPR-Fluorescent protein
18
Q
CRISPR/Cas9 gene editing in people
A
Deletion of CCR5, which encodes protein that allows HIV to enter cells
Leber Congenital Amaurosis (LCA) - loss of eyesight
Sickle Cell Disease
19
Q
CRISPR/Cas9 Genome editing
A
plants/livestock
Modified calves to have their DNA edited so that they don’t grow horns
20
Q
DNA DSC repair and gene editing
A
Various targeting/cutting methods produce DSBs at specific sites in DNA
Those need to be repaired, and typically NHEJ dominates
NHEJ can disrupt a gene, but not incorporate specific mutations
HR can use a provided template to incorporate specific mutations
21
Q
Condensing DNA
A
Through action of proteins:
Histones
Nonhistone chromosomal proteins
DNA-protein complex is called chromatin
22
Q
Stages of DNA condensation
A
10 nm fiber, 30 nm fiber, looped domains, condensed chromatin, metaphase chromosomes
23
Q
10 nm fiber (DNA condensation)
A
Also known as “beads on a string” structure.
Nucleosome core particles (histone octamers with wrapped DNA) connected by linker DNA.
24
Q
30 nm fiber (DNA condensation)
A
Further compaction of nucleosomes into a higher-order structure.
Interactions between histone tails and linker DNA contribute to folding.
25
Looped domains (DNA condensation)
30 nm fibers organized into looped domains anchored by protein complexes.
Each loop contains multiple nucleosomes and is stabilized by proteins like cohesin.
26
Condensed chromatin (DNA condensation)
Further compaction of looped domains through additional folding and looping.
Chromatin loops may interact with each other to form higher-order structures.
27
Metaphase chromosomes (DNA condensaton)
Maximum condensation achieved during cell division.
Chromosomes appear as highly condensed, distinct entities visible under a microscope.
28
Histone features
Small (just over 100 amino acids)
Basic
Highly conserved (just a few amino acid changes between human and pea
The N-terminal tails are the sites of covalent modifications that regulate nucleosomes
29
Histone octamer formation
Eight histone proteins (two copies each of H2A, H2B, H3, and H4) form a histone octamer.
Histone fold domains interact within and between histone proteins.
Octamer formation occurs through extensive histone-histone interactions and stabilization of the structure.
30
Histone fold
Structural motif found in histone proteins.
Consists of three alpha helices connected by two loops.
Facilitates histone-histone interactions and DNA wrapping.
31
Histone Octamer Path of DNA
DNA wraps around the histone octamer in a left-handed superhelical turn.
Approximately 1.65 turns of DNA wrap around each histone octamer.
Nucleosome core particle consists of ~147 base pairs of DNA wrapped around the histone octamer.
32
how DNAse I was used to reveal various features of chromatin
Digest chromatin with a non-specific exonuclease like DNase I to cleave between the nucleosomes. Treatment with a high salt concentration will dissociate the octamer.
By digestion chromatin with a low concentration of DNase I, we can see a ladder of nucleosomes. Here, we are not cutting between every pair of nucleosomes so we have mononucleosomes at 200 nte, dinucleosomes at 400 nte, etc.
33
how DNA footprinting can be used to show where proteins bind on DNA
Identifies protein-bound DNA regions.
Relies on differential DNA cleavage due to protein binding.
34
ChIP-seq experiment (chromatin immunoprecipitation followed by DNA sequencing)
Chromatin immunoprecipitation (ChIP):
Cross-link DNA and proteins in cells.
Fragment chromatin and immunoprecipitate protein of interest along with bound DNA fragments using specific antibodies.
DNA sequencing:
Purify and amplify immunoprecipitated DNA fragments.
Data analysis:
Identify enriched regions of DNA binding for the protein of interest.
Interpretation:
Determine genomic locations where the protein binds, providing insights into its regulatory roles.
35
H1
Acts as a linker histone in chromatin structure.
Binds to the DNA between nucleosomes, facilitating compaction of chromatin into higher-order structures.
Plays a role in stabilizing nucleosome positioning and regulating accessibility of DNA for transcription and other nuclear processes.
Involved in the regulation of gene expression, chromatin condensation during cell division, and DNA repair processes.
36
Chromatin remodeling
Process of altering chromatin structure to regulate access to DNA.
Involves ATP-dependent remodeling complexes that slide, eject, or restructure nucleosomes.
Facilitates transcription, DNA repair, and other nuclear processes by modulating DNA accessibility.
Essential for gene regulation and cellular differentiation.
37
Histone variants
Variants of core histones with distinct amino acid sequences.
Replace canonical histones in nucleosomes, altering chromatin structure and function.
Examples include H2A.Z, H3.3, and macroH2A, each with specific roles in transcriptional regulation, DNA repair, and genome stability.
Contribute to chromatin dynamics, epigenetic regulation, and cellular differentiation.
38
Histone modifications
Chemical alterations to histone proteins, including acetylation, methylation, phosphorylation, and ubiquitination.
Regulate chromatin structure and function by influencing DNA accessibility, transcription, and other nuclear processes.
Mediated by histone-modifying enzymes, such as histone acetyltransferases (HATs), histone methyltransferases (HMTs), and histone kinases.
39
Effects of histone modification
Acetylation, Methylation, Phosphorylation, Ubiquitination
40
Acetylation
Generally associated with gene activation by loosening chromatin structure.
41
Methylation
Can be linked to both gene activation and repression depending on the lysine residue and methylation status.
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Phosphorylation
Involved in transcriptional activation, DNA repair, and mitosis.
43
Ubiquitination
Associated with gene silencing and DNA damage response.
44
Histone code
Concept proposing that specific combinations of histone modifications act as a molecular language to regulate chromatin states and gene expression.
Patterns of histone modifications serve as signals for recruitment of effector proteins and chromatin remodeling complexes.
Provides a dynamic and reversible mechanism for epigenetic regulation of gene expression and cellular processes.
45
code reading proteins
the protein domain that binds methylated lysine is called a chromodomain or PHD fingers/domains
the binding of other proteins besides histones to the chromatin may act to set up physical placeholders that have the effect of phasing nucleosomes
chromodomains generally have a pocket wherein the methylated lysine can fit
46
Steps in transcription
initiation, elongation, termination
47
initiation
RNA polymerase binds to the promoter region of the DNA molecule, marking the beginning of transcription. This binding forms a transcription initiation complex.
48
elongation
RNA polymerase unwinds the DNA double helix near the transcription start site, exposing the template strand.
The enzyme then synthesizes an RNA molecule complementary to the template DNA strand by adding complementary RNA nucleotides.
As RNA polymerase moves along the DNA template, it continues to unwind the DNA ahead of it and synthesize RNA behind it.
49
Termination
Transcription continues until RNA polymerase reaches a termination signal in the DNA sequence.
In prokaryotes, termination often involves the formation of a hairpin loop in the RNA transcript followed by the dissociation of RNA polymerase from the DNA template.
In eukaryotes, termination is more complex and involves specific proteins and sequences that signal the end of transcription.
50
sigma factor
Sigma factors initiate bacterial transcription by binding to promoters and directing RNA polymerase.
51
transcription termination
1. Rho-dependent termination
Requires ‘Rho’ protein
2. Intrinsic (Rho-independent) termination
No protein required
52
the significance the -35 and -10 boxes, and know the consensus sequence of the -10 box
Sigma factor provides specificity to promoter recognition by RNA polymerase
- sigma factor directly contacts the DNA at the promoter site in the -35 and -10 regions
53
details of sigma factor binding to DNA
-alternative sigma factors associate with the same core RNA polymerase, but recognize distinct promoters
54
how alternate sigmas turn on other sets of genes
Alternate sigma factors, like σ^32 in E. coli, regulate gene expression under stress.
σ^32 activates heat shock genes, aiding protein folding and degradation during heat stress.
This enables bacteria to adapt swiftly to environmental changes, ensuring survival.
55
ways in which a ligand can work with a DNA-binding protein in positive regulation
A ligand can positively regulate gene expression by a DNA-binding protein's affinity for DNA, facilitating transcription initiation.
Ligand-induced conformational changes can modulate DNA-binding affinity, impacting gene regulation.
56
ways in which a ligand can work with a DNA-binding protein in negative regulation
In negative regulation, ligand binding inhibits DNA binding, repressing gene expression.
Ligand-induced conformational changes can modulate DNA-binding affinity, impacting gene regulation.
57
lac operon: structure, protein components, and how it works
Both positive AND negative regulation take place.
When glucose is low, cAMP levels rise and bind/activate binding of CAP protein at the promoter.
Allosteric: Required to improve “fit” of sigma factor with promoter sequence
58
When lactose is low,
the repressor has no lactose bound and BINDS to the operator to block polymerase progression.
59
When lactose is high,
lactose binds the repressor and causes an allosteric change, making it unable to bind the operator.
60
When glucose is low,
cAMP levels rise and bind/activate binding of CAP protein at the promoter.
61
Understand the different phenotypes of lac constitutive mutants in merodiploid cells
In merodiploid cells, lac constitutive mutants show varying phenotypes due to mutations in the lac repressor gene.
Partial constitutive mutants exhibit low-level expression, while fully constitutive mutants show unregulated high-level expression.
Super-repressors repress the lac operon even in the presence of lactose.
62
the function of catabolite repression, cAMP, and CAP
Genes required for the metabolism of alternative carbon sources in E. coli are also subject to catabolite repression: their transcription does not occur when glucose is available.
– E. coli grown in the presence of lactose AND glucose do not transcribe the lac operon.
– Glucose is used in preference to any other carbon source; only when glucose is depleted are other compounds metabolized.
– Catabolite repression is controlled by the Catabolite Activator Protein (CAP) and cAMP, which binds to CAP.
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E. coli grown in the presence of lactose AND glucose
do not transcribe the lac operon.
64
Catabolite repression is controlled by
the Catabolite Activator Protein (CAP) and cAMP, which binds to CAP.
65
trp repressor function
An inducible system: responds to the environment.
* When trp is high, trp binds the trp repressor protein to produce a structure that CAN bind the DNA via helix turn helix (allosteric effect on the structure of the repressor protein) – BLOCKS EXPRESSION.
* When trp is low—helix turn helix structure is different because there is no tryptophan bound to the protein, repressor protein DOES NOT bind DNA – EXPRESSION ALLOWED.
66
When trp is high, (trp operon)
trp binds the trp repressor protein to produce a structure that CAN bind the DNA via helix turn helix (allosteric effect on the structure of the repressor protein) – BLOCKS EXPRESSION.
67
When trp is low (trp operon)
helix turn helix structure is different because there is no tryptophan bound to the protein, repressor protein DOES NOT bind DNA – EXPRESSION ALLOWED.
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Attenuation
A novel mechanism of regulation of the trp Operon
Attenuation: a second regulatory mechanism
- sensor of cellular charged tRNA concentration
low typtophan levels: no attenuation
high tryptophan levels cause attenuation
no translation causes attenuation
69
low tryptophan levels
no attenuation
70
high tryptophan levels
cause attenuation
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no translation (trp operon)
causes attentuation
72
roles of LexA and RecA in SOS response
In the SOS response, LexA acts as a repressor, regulating DNA repair gene expression.
When DNA damage occurs, RecA is activated, promoting LexA cleavage. This allows for the upregulation of DNA repair pathways.
73
riboswitches
Riboswitches, found in mRNA UTRs, regulate gene expression by binding ligands.
Ligand binding induces a conformational change, modulating gene expression levels.
This mechanism allows cells to sense small molecule concentrations and adjust gene expression accordingly.
74
RNA-seq experiment can show which genes are turned on in which cells
RNA-seq measures gene expression by sequencing RNA molecules.
After sequencing and alignment, statistical analysis identifies active genes in different cell types or conditions.
75
prokaryotic RNA polymerase enzymes (subunits)
is composed of five subunits in its core enzyme: two α subunits, one β subunit, one β' subunit, and one ω subunit.
Additionally, it associates with a sigma (σ) subunit to form the holoenzyme, which recognizes promoter sequences and initiates transcription.
76
eukaryotic RNA polymerase enzymes (subunits)
eukaryotic RNA polymerase II is more complex, consisting of 12 subunits.
The largest subunit, Rpb1, houses the catalytic center responsible for RNA synthesis, while Rpb2 contributes to RNA binding.
Other subunits play roles in stabilizing the enzyme, facilitating initiation, elongation, and termination of transcription.
77
general features of eukaryotic pol II promoters, including the consensus TATA box sequence
Eukaryotic RNA polymerases require additional components for recognition of their promoter and initiation
* Eukaryotic RNA Pol:
Core promoter region with essential elements like the TATA box (consensus sequence "TATAAA").
Initiator (Inr) element near the transcription start site.
Proximal promoter elements such as the downstream promoter element (DPE) and motif ten element (MTE).
Regulatory elements like enhancers and silencers.
Transcription factor binding sites for precise gene expression control.
78
Prokaryotic RNA pol
– Requires sigma factor (one at a time) for recognition of promoters and initiation of transcription
79
Eukaryotic RNA pol
– Need something similar: General Transcription Factors (GTFs)
– Multiple GTFs are required (unlike the single sigma factor)
– Some of them recognize conserved sequence elements in the promoter.
– We will focus on those used by RNA Pol II for gene expression
80
general transcription factors
TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH
81
TFIID
Binds to the TATA box, positioning RNA polymerase II (Pol II).
Recognizes other DNA sequences near the transcription start point; regulates DNA-binding by TBP
TBP subunit, TAF subunits, TFIIA
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TFIIA
Stabilizes TFIID binding and prevents non-specific DNA interactions.
83
TFIIB
Recognizes BRE element in promoters
Helps to position Pol II at the transcription start site
84
TFIIF
stabilizes RNA polymerase interaction with TBP and TFIIB
helps attract TIIE and TFIIH
85
TFIIE
attracts and regulates TFIIH
86
TFIIH
Unwinds DNA at the transcription start point, phosphorylates Ser5 of the RNA pol CTD
Releases RNA polymerase from the promoter
87
the composition of TFIID and the role of TB
TFIID is composed of TATA-binding protein (TBP) and TBP-associated factors (TAFs).
TBP recognizes and binds to the TATA box within gene promoters, positioning TFIID and RNA polymerase II for transcription initiation.
TAFs stabilize TFIID and contribute to transcriptional regulation.
88
the steps in the assembly of a transcription initiation complex
a. TFIID binds to the TATA box in the promoter region, facilitated by TBP.
b. TFIIA stabilizes the TFIID-DNA interaction and prevents non-specific binding.
c. TFIIB binds to the BRE element upstream of the TATA box, positioning RNA Pol II at the transcription start site.
d. TFIIF stabilizes the preinitiation complex.
e. TFIIE recruits TFIIH to the complex.
f. TFIIH unwinds DNA at the transcription start site and phosphorylates the C-terminal domain (CTD) of RNA Pol II, activating it for transcription initiation.
89
the carboxy terminal domain of RNA pol II and its phosphorylation
The CTD is a flexible and unstructured domain located at the C-terminus of RNA Pol II.
It consists of multiple repeats of a heptapeptide sequence (Tyr-Ser-Pro-Thr-Ser-Pro-Ser).
Phosphorylation of specific serine residues within the CTD by TFIIH regulates various stages of transcription, including initiation, elongation, and termination.
90
enhancer elements
Enhancers are DNA sequences that can be located upstream, downstream, or within introns of target genes.
They are typically bound by transcription factors, which can interact with the basal transcription machinery to increase transcriptional activity.
Enhancers can function over long distances and in an orientation-independent manner.
They often contain specific DNA-binding motifs for transcription factors and can loop to bring distal enhancers into close proximity to the promoter region, facilitating transcriptional activation
91
DNA binding domains
DNA binding domains are regions within transcription factors that specifically recognize and bind to DNA sequences.
These domains often contain conserved amino acid motifs or structural motifs that allow them to interact with the double-stranded DNA in a sequence-specific manner.
Examples of DNA binding domains include the helix-turn-helix motif, zinc finger motif, leucine zipper motif, and homeodomain.
92
Homeodomain
is a DNA binding domain found in a class of transcription factors known as homeobox proteins.
It consists of approximately 60 amino acids and forms a characteristic three-dimensional structure, including three alpha helices.
typically bind to DNA sequences known as homeoboxes, which are specific DNA sequences involved in developmental gene regulation.
93
Dimerization of transcription factors
Many transcription factors function as dimers, meaning they consist of two subunits that come together to form an active complex.
Dimerization can occur through various mechanisms, such as the formation of homodimers (two identical subunits) or heterodimers (two different subunits).
Dimerization can alter the function of transcription factors by changing their DNA binding specificity, affinity, or ability to recruit co-factors or other regulatory proteins
94
how to identify functional domains in a protein
Functional domains in proteins can be identified through various bioinformatics and experimental approaches.
Bioinformatics tools can analyze protein sequences to predict the presence of conserved domains, motifs, or structural features associated with specific functions.
Domain mapping studies, such as deletion or mutagenesis analysis, can help identify regions of a protein that are essential for its biological activity or interaction with other molecules.
95
coactivators
- Coactivators interact with transcription factors and the transcriptional machinery.
- They enhance gene expression by bridging interactions between factors.
- Coactivators include histone acetyltransferases (HATs).
- HATs acetylate histone proteins, relaxing chromatin structure for transcription.
- Coactivators may mediate other chromatin modifications.
96
mediators
Mediator is a multisubunit protein complex that acts as a bridge between transcription factors, the basal transcription machinery, and RNA polymerase II.
It facilitates communication between transcriptional activators bound to enhancers and the preinitiation complex at the promoter region, promoting transcription initiation.
Mediator consists of multiple subunits, each with specific functions in transcriptional regulation, including bridging interactions between regulatory proteins, modifying chromatin structure, and recruiting RNA polymerase II.
Mediator: Bridges interactions between transcription factors and RNA polymerase II.
97
three mechanisms of transcriptional repression
direct repression by transcription factors, chromatin remodeling and histone modifications, and RNA mediated repression
98
direct repression by transcription factors
Transcription factors bind to specific DNA sequences (silencer elements) near gene promoters.
Repressors recruit co-repressors or chromatin-modifying complexes to inhibit transcription initiation.
Repressed chromatin state blocks access of RNA polymerase and transcriptional machinery to gene promoters.
99
Chromatin remodeling and histone modifications
Histone deacetylases (HDACs) remove acetyl groups from histone tails, leading to chromatin condensation and transcriptional repression.
Histone methyltransferases (HMTs) add methyl groups to histones, promoting repressive chromatin states.
ATP-dependent chromatin remodeling complexes alter nucleosome positioning or density, restricting access to DNA.
100
RNA mediated repression
Non-coding RNAs (e.g., microRNAs, long non-coding RNAs) bind to complementary sequences in target mRNAs.
RNA-RNA interactions inhibit translation or promote mRNA degradation through RNA interference (RNAi) pathways.
RNA-binding proteins can also sequester or destabilize target mRNAs, preventing their translation.
101
how the activity of transcription factors can be regulated
Post-translational modifications. protein-protein interactions, cellular signaling pathways
102
Post-translational modifications
Phosphorylation, acetylation, methylation, and other modifications can alter the activity, stability, subcellular localization, and DNA-binding affinity of transcription factors, affecting their ability to regulate gene expression.
103
protein-protein interactions
Transcription factors can interact with coactivators or corepressors, which modulate their activity by influencing their recruitment to target genes, DNA-binding specificity, or transcriptional activation/repression.
104
Cellular signaling pathways
Extracellular signals activate intracellular signaling cascades that regulate the activity of transcription factors through phosphorylation, proteolytic cleavage, or changes in subcellular localization, allowing cells to respond dynamically to environmental cues and physiological changes.
105
Understand the carboxy terminal domain of RNA pol II, its heptapeptide repeat
Located at the C-terminus of RNA Pol II.
Consists of multiple repeats of a heptapeptide sequence (Tyr-Ser-Pro-Thr-Ser-Pro-Ser)
106
Know the phosphorylation of the CTD by TFIIH causes the switch from initiation to elongation complex
TFIIH is a transcription factor complex.
Phosphorylates serine residues within the CTD.
Causes a conformational change in RNA Pol II, switching from initiation to elongation complex.
107
insulators
DNA sequences that block or insulate the effect of enhancers or silencers on neighboring genes.
Prevent inappropriate activation or repression of nearby genes by enhancers or silencers.
Form boundary elements that maintain the integrity of chromatin domains and regulate gene expression patterns.
108
control of expression of all genes discussed in class
Transcription factors: Bind to specific DNA sequences to activate or repress gene transcription.
Enhancers/silencers: DNA elements that enhance or suppress gene expression, respectively.
Promoters: DNA sequences where RNA polymerase binds to initiate transcription.
RNA interference (RNAi): Post-transcriptional gene silencing by small RNAs (siRNA or miRNA).
109
the role of chromatin modification on gene expression
Histone acetylation: Opens chromatin structure, facilitating gene transcription.
Histone methylation: Can activate or repress gene expression depending on the specific lysine residue modified.
Histone phosphorylation: Associated with transcriptional activation and elongation.
DNA methylation: Often associated with gene silencing by inhibiting transcription factor binding.
110
what other factors can ‘ride along’ on the phosphorylated CTD
capping enzymes, splicing factors, polyadenylation factors
111
capping enzymes
involved in mRNA capping process
112
splicing factors
facillitate pre-mRNA splicing
113
Polyadenylation factors
participate in mRNA polyadenylation
114
Know the structure of the cap and the enzymes that put it on
mRNA cap: Consists of a 7-methylguanosine residue linked to the 5' end of mRNA via a 5'-5' triphosphate bridge.
Enzymes: capping enzymes, including capping enzyme (CE), RNA triphosphatase (RTPase), and guanylyltransferase (GTase).
115
the steps in polyadenylation and the enzymes responsible
Cleavage: Endonuclease cleaves pre-mRNA downstream of the polyadenylation signal (AAUAAA).
Polyadenylation: Poly(A) polymerase (PAP) adds a string of adenine nucleotides (poly(A) tail) to the 3' end
.
Polyadenylation factors: Includes cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and poly(A) polymerase (PAP).
116
Understand the 3’ end processing of histone mRNAs: stem/loop, cleavage site, HDE
Stem/loop structure: Forms at the 3' end of histone pre-mRNA, serving as a recognition site.
Cleavage site: Located upstream of the stem/loop structure where endonuclease cleaves.
Histone downstream element (HDE): RNA element downstream of cleavage site that binds stem-loop binding protein (SLBP), stabilizing the stem/loop structure.
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Stem/loop structure
Forms at the 3' end of histone pre-mRNA, serving as a recognition site.
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Cleavage site
Located upstream of the stem/loop structure where endonuclease cleaves.
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Histone downstream element (HDE)
RNA element downstream of cleavage site that binds stem-loop binding protein (SLBP), stabilizing the stem/loop structure.
120
U7
Associates with U7-specific protein (Lsm11) to form the U7 small nuclear ribonucleoprotein particle (snRNP).
U7 snRNP binds to the histone pre-mRNA 3' end and directs cleavage at the cleavage site.
Facilitates histone mRNA 3' end processing and ensures proper maturation.
121
the roles of the cap and the poly(A) tail in translation and the proteins involved
Cap structure: Facilitates mRNA recognition by ribosomes and enhances translation initiation.
Poly(A) tail: Enhances mRNA stability, facilitates ribosome binding, and promotes translation initiation.
Proteins involved: eIF4E binds to the cap, while poly(A)-binding proteins (PABPs) bind to the poly(A) tail, both promoting translation initiation.
122
the R-looping experiment with Adenovirus DNA and mRNA the revealed the existence of introns
Hybridization of labeled mRNA to DNA forms RNA-DNA hybrids (R-loops).
R-looping revealed unhybridized regions in DNA, suggesting the presence of introns.
Presence of unhybridized regions indicated that introns were spliced out from pre-mRNA.
123
explain the conserved sequences at the ends and in the center of introns
5' splice site: Consists of GU nucleotides at the 5' end of introns.
Branch point sequence: Contains an adenine nucleotide located near the branch site within the intron.
3' splice site: Consists of AG nucleotides at the 3' end of introns.
These sequences are recognized by the spliceosome during splicing and are essential for intron removal.
124
Know two protein complexes that bind the branch site in pre-mRNA splicing: BBP and U2AF
BBP and U2AF
125
BBP
branch point binding protein): Recognizes and binds to the branch site sequence in pre-mRNA.
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U2AF
(U2 auxiliary factor): Consists of two subunits, U2AF65 and U2AF35, which also bind to the branch site region.
127
the 2 step reaction mechanism including attack by the 2’ OH of the branch site
recognition and cleavage of 5' splice site
Attack by 2' OH of the branch site
128
recognition and cleavage of 5' splice site
U1 snRNP binds to the 5' splice site, aided by factos like SF1 and BBP
TNA duplex forms between U1 snRNA and 5' splice site
cleavage occurs at 5' splice site, releasing 5' exon and forming a lariat intermediate
129
Attack by 3' OH of the branch site
U2 snRNP, along with U2AF, binds to the branch
Branch site adenosine nulceophile attacks the phosphate at the 5' splice site
resulting in the formation of lariat structure and joining of the 5' and 3' exons
130
Structure of the lariat intermediate
Formed during pre-mRNA splicing.
Consists of a looped structure where the 5' end of the intron is joined to the branch site via a 2'–5' phosphodiester bond.
The 3' end of the intron is released as a lariat-shaped molecule.
131
the properties of small nuclear RNAs
Short non-coding RNA molecules found in the nucleus.
Combine with proteins to form small nuclear ribonucleoprotein particles (snRNPs).
Essential components of the spliceosome, involved in pre-mRNA splicing.
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U1 snRNA
binds to 5' splice site, initiating spliceosome assembly
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U2 snRNA
binds to the branch site, aiding in formation of the catalytic center
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U4 snRNA
initially binds to U6 snRNA, preventing premature activation of U6
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U5 snRNA
stabilizes interactions within the spliceosome and participates in catalysis
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U6 snRNA
participates in catalysis and displaces U1 and U4 snRNAs during spliceosome activation
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how snRNPs assemble onto the pre-mRNA to form a mature spliceosome
U1 snRNP binds to the 5' splice site.
U2 snRNP binds to the branch site.
U4/U6.U5 tri-snRNP complex associates with the pre-spliceosome.
Additional factors facilitate the formation of the mature spliceosome.
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the interactions at the 5’ splice site and the branch site
U1 snRNP interacts with the 5' splice site via base pairing between U1 snRNA and the pre-mRNA.
U2 snRNP recognizes and binds to the branch site through base pairing interactions with U2 snRNA.
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Group I self splicing introns
catalyze their own excision from pre-mRNA through two step splicing mechanism involving internal guanosine as a cofactor
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Group II self splicing introns
also cayalyze their own excision but use a different mechanism than group I introns
they have a conserved secondary structure and utilize a bulged adenosine as a nucleophile in the splicing reaction
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how group I introns were discovered
Discovered in Tetrahymena thermophila by Thomas Cech in the 1980s.
Studying the ribosomal RNA (rRNA) of Tetrahymena led to the identification of self-splicing introns.
Observed that the introns catalyzed their own excision from the primary transcript.
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The role of the ‘exogenous G’ in group I splicing
Group I introns require a guanosine cofactor (exogenous G) for splicing.
The guanosine binds to a specific site within the intron RNA, termed the guanosine-binding site (GBS).
The exogenous G participates as a cofactor in the first step of splicing, where it attacks the 5' splice site, initiating intron excision
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The similarities between group II and pre-mRNA introns
Both are found in eukaryotic organisms.
Both undergo splicing to remove intronic sequences from the primary transcript.
Both splicing reactions are catalyzed by ribonucleoprotein complexes.
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five different modes of alternative splicing
exon skipping, intron retention, Alternative 5' splice site, alternative 3' splice site, and mutually exclusive exons
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exon skipping
exon is excluded from the final mRNA transcript
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intron retention
intron is retained in the final mRNA transcript
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alternative 5' splice site
different 5' splice sites are used, resulting in alternative exon boundaries
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alternative 3' splice site
different 3' splice sites are used, resulting in alternative exon boundariesm
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mutually exclusive exons
one of two exons is included in the final mRNA transcript, but not both
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the different mRNAs that can be made from the Drosophila DSCAM gene
DSCAM (Down Syndrome Cell Adhesion Molecule) gene in Drosophila undergoes extensive alternative splicing.
mature mRNA:
20 constitutive exons,
4 cassette exons
It can produce thousands of different mRNA isoforms through alternative splicing.
Variability arises from the selection of different combinations of exon 4, 6, 9, and 17.
This diversity in mRNA isoforms contributes to neuronal diversity and axon guidance in Drosophila development.
The resulting protein isoforms play roles in neuronal development and function.
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Regulation of alternative splicing
is regulated by
positive and negative controls
Regulation of alternative splicing is similar to transcriptional regulation.
* Repressor and activator proteins bind to regulatory sites on the
RNA (= silencer and enhancer sequences):
- Intronic and exonic splicing silencers (ISS, ESS)
- Intronic and exonic splicing enhancers (ISE, ESE)
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Negative control of alternative splicing
(no or reduced splicing), a repressor protein binds to the pre-mRNA at a splicing suppressor and blocks splicing, often causing a cryptic splice site to be used.
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Positive control of alternative splicing
(increased splicing), splicing is inefficient unless an activator
protein binds in the region (to a splicing enhancer sequence) to help.
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consequences of alternative splicing
Swapping transactivation domains of transcription factors
* Swapping DNA-binding domains of transcription factors
* Loss of regulation of transcription factors and enzymes (become
constitutive)
* Changes in intracellular localization (membrane-bound vs
cytoplasmic, etc.)
* Loss of enzymatic activity
* Changes in RNA stability [note: alt. splicing affects RNA
properties as well!]
* Changes in RNA localization
* Cause disease
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Aberrant Splicing Causes Disease
Splicing abnormalities such as
exon skipping and intron
retention account for up to 15%
of all inherited diseases.
* Prominent examples are beta-thalassemia, cystic fibrosis,
muscular dystrophies, inherited
cancer predisposition
syndromes, Parkinson’s.
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Know how alternative polyadenylation can alter antibody protein localization
allows antibodies to be displayed on the cell surface
To make the membrane bound antibody:
Remove the yellow intron (and its stop
codon). Now the mRNA includes the light blue exon and its stop codon is used.
This makes the protein (green) bigger, including the membrane-anchoring
hydrophobic peptide.
To make the secreted antibody:
Don’t remove the yellow intron (and its stop
codon). Now the mRNA is shorter and lacks the terminal hydrophobic region, so it
is secreted.
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"exon definition" model
It’s not introns that get marked, but exons. They are marked co transcriptionally by factors that ride along on the RNA pol II
CTD. In this first step, exons are defined.
Relies on interactions between splice sites within the same exon.
Splicing machinery recognizes and defines exons by pairing splice sites within the same exon.
Allows for accurate recognition of exons and exclusion of introns during splicing.
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RNA interference
describes the inhibition of gene
expression by short single-stranded RNAs that bind to target
mRNAs via complementary base pairing.
* These small noncoding RNAs (small ncRNAs) can cause
degradation of the target mRNA or inhibit its translation.
* There are 3 general classes of small ncRNAs that carry out
RNAi:
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3 general classes of small ncRNAs that carry out
RNAi:
microRNAs (miRNAs)
– small interfering RNAs (siRNAs)
– piwi-interacting RNAs (piRNAs)
* The sources and formation of these small ncRNAs differs, but
their mechanisms of action are similar
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RISC complex
(RNA-induced silencing complex): Effector complex of RNAi pathway that mediates target mRNA degradation or translational repression.
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Overview of RNA Interference in Eukaryotes
- mRNA degradation is the primary mechanism in mammals
- translational repression is the primary mechanism in plants and
lower eukaryotes
- heterochromatin formation has been demonstrated primarily in
plants and fission yeast
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Learn the experiment that identified the active component in RNAi
Andrew Fire and Craig Mello's experiment in Caenorhabditis elegans in 1998.
Demonstrated that double-stranded RNA (dsRNA) triggers potent and specific gene silencing.
Discovered that RNAi is mediated by short interfering RNAs (siRNAs) derived from dsRNA.
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Dicer
RNAse III endonuclease that processes precursor miRNAs into mature miRNAs, and also generates siRNAs from long dsRNA.
"Dicing"
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drosha
Enzyme involved in microRNA (miRNA) biogenesis, cleaving primary miRNA transcripts into precursor miRNAs.
"cropping"
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Argonaute
Key component of RISC complex, responsible for binding to small RNAs and guiding RISC to target mRNAs for silencing or degradation
