Exam 2 Review Flashcards
Describe how to identify membrane-spanning protein in genomic sequence
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
Gene knock-out
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.
Gene knock-in
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.
Meganucleases
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
Zinc finger motif
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
Zinc finger nucleases (ZFNs)
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.
Structure of zinc finger domains
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.
Controlling sequence-specificity (zinc finger)
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.
Transcription activator-like effectors (TALEs)
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.
TALENs (TALE nucleases)
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.
Features and Uses of TALEs and TALENs
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.
Describe how bacteria use CRISPR/Cas-9 to fight viruses
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.
Describe how to use CRISPR/Cas-9 to cut any DNA sequence
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.
other uses of CRISPR/Cas-9
multiplexing, gene editing, genome editing
CRISPR/Cas-9 advantages over other systems
DNA recognition depend on sgRNA instead of protein domains
Very easy to design/clone sgRNAs
Highly specific
Homozygous targeting
Multplexing
multiplexing
Gene targeting with ZFN and TALENs require multiple rounds of editing for multiple genes
CRISPR/Cas9 can be multiplexed-simply adding multiple sgRNAs
CRISPR/Cas9 applications
Gene expression repression (inhibition) = CRISPRi
Gene expression activation = CRISPRa
Painting the genome CRISPR-Fluorescent protein
CRISPR/Cas9 gene editing in people
Deletion of CCR5, which encodes protein that allows HIV to enter cells
Leber Congenital Amaurosis (LCA) - loss of eyesight
Sickle Cell Disease
CRISPR/Cas9 Genome editing
plants/livestock
Modified calves to have their DNA edited so that they don’t grow horns
DNA DSC repair and gene editing
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
Condensing DNA
Through action of proteins:
Histones
Nonhistone chromosomal proteins
DNA-protein complex is called chromatin
Stages of DNA condensation
10 nm fiber, 30 nm fiber, looped domains, condensed chromatin, metaphase chromosomes
10 nm fiber (DNA condensation)
Also known as “beads on a string” structure.
Nucleosome core particles (histone octamers with wrapped DNA) connected by linker DNA.
30 nm fiber (DNA condensation)
Further compaction of nucleosomes into a higher-order structure.
Interactions between histone tails and linker DNA contribute to folding.
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.
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.
Metaphase chromosomes (DNA condensaton)
Maximum condensation achieved during cell division.
Chromosomes appear as highly condensed, distinct entities visible under a microscope.
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
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.
Histone fold
Structural motif found in histone proteins.
Consists of three alpha helices connected by two loops.
Facilitates histone-histone interactions and DNA wrapping.
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.
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.
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.
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.
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.
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.
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.
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.
Effects of histone modification
Acetylation, Methylation, Phosphorylation, Ubiquitination
Acetylation
Generally associated with gene activation by loosening chromatin structure.
Methylation
Can be linked to both gene activation and repression depending on the lysine residue and methylation status.
Phosphorylation
Involved in transcriptional activation, DNA repair, and mitosis.
Ubiquitination
Associated with gene silencing and DNA damage response.
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.
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
Steps in transcription
initiation, elongation, termination
initiation
RNA polymerase binds to the promoter region of the DNA molecule, marking the beginning of transcription. This binding forms a transcription initiation complex.
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.
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.
sigma factor
Sigma factors initiate bacterial transcription by binding to promoters and directing RNA polymerase.
transcription termination
- Rho-dependent termination
Requires ‘Rho’ protein - Intrinsic (Rho-independent) termination
No protein required
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
details of sigma factor binding to DNA
-alternative sigma factors associate with the same core RNA polymerase, but recognize distinct promoters
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.
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.
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.
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
When lactose is low,
the repressor has no lactose bound and BINDS to the operator to block polymerase progression.
When lactose is high,
lactose binds the repressor and causes an allosteric change, making it unable to bind the operator.
When glucose is low,
cAMP levels rise and bind/activate binding of CAP protein at the promoter.
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.
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.
E. coli grown in the presence of lactose AND glucose
do not transcribe the lac operon.
Catabolite repression is controlled by
the Catabolite Activator Protein (CAP) and cAMP, which binds to CAP.
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.
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.
