SRP, SR Location, GTPase Domain and Sec61

SRP/SR Location and GTPase Domain

• Signal recognition particle (SRP) is located in the cytoplasm and on the inner membrane of the SR (smooth reticulum).
• SR receptor is located on the inner SR membrane.
• Both SRP and SR contain NG GTPase domains.
• Before GTP binding, the signal sequence must bind the RNC-SRP targeting complex, which then allows SRP to bind GTP
• SR needs to be close to an empty Sec61 complex before it too binds GTP.
• GTP hydrolysis requires NG-NG twin formation between Ffh (in SRP) and FtsY (in SR), combined with relocation of the twin to the distal end of the 4.5S RNA.
• GTPase activation is triggered by a flipped-out nucleotide at the distal end of SRP-RNA.

Sec61 Structure and Membrane Transport

• Sec61 consists of a central "plug" which acts as a seal that prevents diffusion of both proteins and ions.
• Sec61 forms an hourglass-shaped aqueous funnel with a pore ring at the central constriction.
• The Sec61 complex has a lateral ("Side") gate.
• Type 1 and type 2 transmembrane protein (TM) co-translational translocation is mediated by the Sec-SRP/SR pathway.
• Longer, more hydrophobic TMs favor type I insertion.
• A rapidly folding domain flanking the TM at the N-terminus favors type II insertion.
• Internal TMs following a translocated part favor type I insertion.
• In bacteria, a charged N-terminus favors type II according to the "positive inside rule".
• Tail-anchored TM protein post-translational translocation on the ER is mediated by TRC40/Get3 and GET1/GET.

Brownian Ratchet vs. Power Stroke Model

• Power stroke model uses the finger domain moving up and down during ATP hydrolysis to push the polypeptide into the SecY channel.
• The Y at the loop’s tip contacts and drags the polypeptide chain, converting ATP into mechanical energy.
• The Brownian ratchet model: In Sec (61, 62/63) and TIM systems, Brownian oscillations result in forward and backward movements of unfolded polypeptide segments in the translocation channel.
• Upon ATP hydrolysis, the peptide-binding pocket of Hsp70 closes around the incoming polypeptide which prevents backsliding and refolding of the preprotein at the cis side.

ATP-Dependent Chaperones

• Trigger factor is a ribosome-bound chaperone associated with cotranslation and has no ATPase activity.
• DnaK possesses a polypeptide-binding domain plus an ATP-binding domain.
• DnaJ (Hsp40) delivers unfolded peptides to DnaK.
• DnaK (Hsp70) binds peptides loosely when ATP-bound (open state), but tightly after ATP hydrolysis (closed state).
• GrpE promotes ADP dissociation; ATP rebinding dissociates GrpE and the peptide.
• GroEL/ES are post-translational chaperones, with GroEL exhibiting ATPase activity.

Protein Stability Factors

• Hydrophobic effect and hydrogen bonds are major stabilizing contributors to protein structure.
• Conformational entropy is a major destabilizing factor.
• Hydrogen bonds contribute to secondary structure stability, primarily on the interior of the protein.

Methods to Monitor Protein Characteristics

• Secondary structure monitored using infrared spectroscopy and circular dichroism (CD).
• Local characteristics are monitored using fluorescence spectroscopy and fluorescence resonance energy transfer (FRET).
• Protein dynamics are monitored using NMR and hydrogen/deuterium-exchange (HDX) combined with either NMR or mass spectrometry.
• Single molecule folding techniques include single-molecule FRET (smFRET) and optical tweezers force microscopy.

Protein Folding Models

• Framework models involve: initial formation of secondary structures and then:

• Diffusion-collision where elements of secondary structure diffuse until they collide. OR

• Nucleation-propagation, where neighboring residues form native-like secondary structures that act as a nucleus.

• Hydrophobic collapse model includes:

• Collapse to a compact bundle.

• Finding native-like tertiary interactions ("Nucleus").

• Formation of native secondary structure.

IRE1 Pathway

• The IRE1 pathway that is activated as part of the unfolded protein response (UPR) in the endoplasmic reticulum.
• When misfolded proteins accumulate in the ER, IRE1 (ER membrane kinase and endoribonuclease) dimerizes and autophosphorylates.
• Autophosphorylation activates its RNase activity.
• This activation causes unconventional splicing of XBP1 mRNA, resulting in the generation of a transcription factor called XBP1s.
• XBP1s upregulates genes involved in protein folding, secretion, and degradation.
• The upregulation works to restore ER homeostasis.

N-Glycosylation

• During N-linked glycosylation, the sequence Asn-X-Ser/Thr is glycosylated, where X is any amino acid except proline.
• Calnexin and calreticulin bind incompletely folded proteins containing one terminal glucose on N-linked oligosaccharides.
• Glucosidase removes the terminal glucose, which releases the protein from calnexin.
• Removal of a central mannose residue causes transfer via vesicle to the cis-Golgi.

Post-Translational Modification (PTM) Summary

• Proteolytic cleavage, such as proprotein maturation of insulin.
• Protein splicing involving inteins.
• Chromophore formation as seen in GFP (green fluorescent protein).
• Glycosylation, lipidation (GPI anchor, acylation, prenylation).
• Hydroxylation of collagen (Hyp, Hyl).
• Carboxylation of prothrombin during calcium 2+ binding.
• Phosphorylation, methylation, and acetylation of histones.
• ADP ribosylation that occurs, for example, due to cholera toxin or pertussis toxin.
• Ubiquitination which causes protein degration.

Protease Classes

• Serine proteases and cysteine proteases form a covalent intermediate during catalysis.
• Aspartyl and metallo proteases do not form a covalent intermediate because they use a general acid-base mechanism for proteolysis.
• Preproteins contain a cleavable N-terminal signal peptide for translocation.
• Proproteins are inactive precursors activated via specific proteolytic cleavage.
• Preproproteins are inactive precursors with both a signal peptide and an inactive pro-region.
• Pre-signals direct proteins to the ER for translocation.
• Pre-pro strategy ensures proteases remain inactive until reaching the correct location, preventing unwanted proteolytic activity.

Apoptosis and Caspases

• Extrinsic apoptosis pathway: Death receptors (Fas, TNFR) recruit FADD, forming the DISC, which activates caspase-8, which then activates executioner caspases.
• Intrinsic apoptosis pathway: Mitochondrial stress releases cytochrome C, which forms the apoptosome with Apaf-1, activating caspase-9, and then executioner caspases.
• Initiator caspases (8, 9) are activated first, triggering executioner caspases.
• Executioner caspases (3, 6, 7) cleave substrates, causing cell death.
• Caspases are cysteine-aspartic proteases that cleave after aspartate residues.

Methods for Stable DNA Integration

• Lentiviral transduction.
• AAV transfection

Diabetes

• Type 1 diabetes is characterized by cell-mediated autoimmune destruction of pancreatic beta cells,.
• Autoantibodies such as those against insulin, glutamic acid decarboxylase (GAD), or protein tyrosine phosphatases IA-2 and IA-2β, serve as indicators of autoimmune response.
• HLA associations: linkages to the HLA-DQA and HLA-DQB genes
• C-peptide levels are typically low or undetectable, reflecting reduced or absent insulin secretion
• Type 1 pathophysiologically works through the autoimmune destruction of insulin-producing beta cells in the pancreas causing little to no insulin production.
• Type 2 pathophysiologically works through insulin resistance in tissues and eventual beta cell dysfunction. Initially there is normal or high production of insulin, but will eventually decrease because of beta cell exhaustion.

Preventing Hyperacute Rejection

• Elimination of carbohydrate xeno-antigens via knockouts of GGTA1, CMAH, and B4GALNT2.
• Expression of human complement pathway regulatory proteins such as CD46, CD55, and CD59.
• Selection of recipients with low pig-reactive antibody levels by getting a negative cross-match.

CRISPR-Cas9

• CRISPR RNA (crRNA): Identifies the DNA sequence target.
• Trans-activating crRNA (tracrRNA): Binds to crRNA, forming a complex that guides Cas9 to the target DNA.
• Single-guide RNA (sgRNA): A fusion of crRNA and tracrRNA into a single molecule.
• Cas9 nuclease: Enzyme that introduces double-stranded breaks in DNA at locations specified by the sgRNA.
• Protospacer Adjacent Motif (PAM): A short DNA sequence following the target, and is necessary for Cas9 binding and cleavage.

Yeast Two-Hybrid Assay

• DNA-binding domain (BD).
• Activation domain (AD).
• Reporter gene.

Hardy-Weinberg Equilibrium

• Allele and genotype frequencies in a population remain constant from generation to generation under the following conditions: no mutation, no gene flow, no selection, infinite population size, and random mating.
• Linkage disequilibrium is when the frequency of a multi-locus haplotype deviates from the expected value based on individual allele frequencies.
• Genetic drift occurs in small populations and can cause allele frequencies to change randomly over time, thus violating the assumption of an infinitely large population.

Cell Differentiation Potency

• Totipotent cells have The ability to differentiate into any cell type. (ex: zygotic.)
• Pluripotent cells (Embryonic) can differentiate into a few types such as:
• Ectoderm: Neurons and skin cells.
• Mesoderm: Muscle cells, and red blood cells.
• Endoderm: Pancreatic cells, and lung cells.
• Multipotent cells are restricted to a single lineage such as: Hematopoietic stem cells, which give rise to various blood cells.

Yeast as a Model Organism

• Yeast grows quickly and can be easily cultured in inexpensive media.
• The well-characterized genome and efficient homologous recombination make yeast highly suitable for targeted genetic modifications and functional studies.
• Many fundamental cellular mechanisms, such as the cell cycle and gene regulation, are conserved between yeast and higher eukaryotes.

GWAS/SNV Details

• A genome-wide association study (GWAS) identifies genetic variations associated with specific traits or diseases.
• Phenotyping: Collect accurate quantitative data (e.g., blood pressure measurements) from a large cohort.
• Genotyping: Genotype participants for SNVs using SNP arrays or sequencing.
• Statistical Analysis: Perform regression analysis for each SNV to test its association with the trait, adjusting for confounders and population stratification.

iPSCs and SCNT

• Yamanaka factors include:
• Oct4 (Oct 3 also correct)
• c-myc
• Sox2
• Klf-4
• iPSCs can be cultured under controlled conditions.
• iPSCs can differentiate into any cell type of the three germ layers by protocols imitating embryonic pathways.
• Somatic cell nuclear transfer (SCNT) is needed.

Model Organism Research

• Paralogs: A pair of genes within one species that is generated by duplication of an ancestral gene.
• Orthologs: A pair of related genes in two different species that is generated by a speciation event.
• Analogs: A pair of unrelated genes in separate species that have similar functions.
• Other factors:
• Tractability in the lab
• Size
• Ease of storage
• Life cycle length
• Generation time (time until ready to reproduce)
• Fertility rate and ease of breeding
• Physical accessibility of features of interest
• Conservation of mechanisms

TFIIH Functions

• TFIIH possesses helicase activity that unwinds the DNA double helix at the transcription start site, allowing RNA polymerase II to access the template strand for transcription initiation.
• TFIIH has kinase activity that phosphorylates the C-terminal domain (CTD) of RNA polymerase II.

Histone Modifications and Function

• Acetylation removes the positive charge of the lysine residue, weakens the interactions with negatively charged nucleosomal DNA and neighboring nucleosomes (e.g., H3K9ac, H3K27ac).
• Methylation is highly site-specific and read by specific reader proteins that determine the functional consequences of methylation.
• H3K4me3 is found at the transcription start site of active genes.
• H3K9me2/3 is associated with heterochromatin.
• H3K27me3 causes polycomb repression.
• Phosphorylation during mitosis is regulated.

DNA Polymerase Fidelity

• Replicative DNA polymerases have 3'→5' exonuclease activity to remove incorrectly incorporated nucleotides.
• Nucleotide Selectivity: During DNA synthesis, DNA polymerases exhibit a strong preference for incorporating nucleotides that correctly base pair with the template strand.
• Higher Processivity:
• Stable Elongation Complex: The β-clamp (bacteria) / PCNA (eukaryotes) keeps the polymerase tightly bound to DNA.

Double-Strand Breaks (DSB)

• Endogenous sources: Head-on and co-directional transcription-replication collisions and reactive oxygen species (ROS) from cellular metabolism.
• Exogenous sources: Ionizing radiation, chemotherapeutic agents, environmental toxins, and indirectly UV radiation through ROS.
• Repair Mechanisms:
• Non-Homologous End Joining (NHEJ): Directly join and ligate broken DNA ends together
• Homologous Recombination (HR): Uses a homologous sequence as a template
• Alternative end-joining (a-EJ): Repair DNA double-strand breaks (DSBs), are initiated by end resection that generates 3′ single strands.

Proximal Pausing

• RNA Pol II enzymes tend to pause following promoter escape in some genes.
• Pausing allows establishment of permissive chromatin, rapid activation of genes,synchronous activation of genes, and the integration of signals.
• Pausing is therefore particularly enriched in signal-regulated pathways.
• Pausing is induced by the protein NELF.
• Release of paused Pol II requires the kinase CDK9 which phosphorylates NELF, DSIF, and S2 of the Pol II CTD.

BER-NER Mechanisms

• Base excision repair (BER) addresses non-helix-distorting base lesions.
• DNA glycosylases recognize and remove the damaged base, creating an abasic (AP) site.
• AP endonucleases then cleave the DNA backbone.
• DNA polymerase inserts the correct nucleotide, and DNA ligase seals the nick.
• Nucleotide excision repair (NER) targets bulky, helix-distorting lesions (caused by UV radiation).
• Begins with damage recognition, followed by dual incisions bracketing the lesion.
• The excised oligonucleotide is removed, and the resulting gap is filled by DNA polymerase with DNA ligase then sealing the remaining nick.
• XPC (xeroderma pigmentosum, complementation group C) detects DNA damage by recognizing distortions in the.

Phase Separation

• Transcription condensates are dynamic, membraneless nuclear compartments that form through liquid-liquid phase separation.
• Weak, multivalent interactions concentrated in transcription factors mediate phase separation.
• Mediator forms condensates and in cells Pol II CTDs domain is also integrated into these condensates.
• Localized enrichment facilitates the rapid assembly and activation of the transcriptional machinery to enhance gene expression efficiency and helps organize chromatin.
• Displays liquid-like properties that allow them to respond swiftly to changes in the cellular environment.

Nucleosome Landscape

• Nucleosome landscape analysis examines the positioning and occupancy of across the genome, providing insights into chromatin structure and gene regulation.
• NFR stands for Nucleosome Free Region.

Mediator Function

• Mediator acts as a bridge between transcription factors and RNA polymerase II and stimulates PIC assembly.
• Contains a conserved core with modules that interact with Pol II, TFIIB, and TFIIH.
• Mediator stimulates phosphorylation of Pol II by the TFIIH kinase subunit CDK7.
• CDK7 phosphorylates the CTD on Ser5, which promotes the transition from initiation to elongation.
• Plays a role in transcription regulation and integrating signals to fine-tune gene expression.

Cohesin/Condensin (SMCs)

• Cohesin Function: Is a protein complex composed of Structural Maintenance of Chromosomes (SMC) proteins (SMC1 and SMC3) that are along with non-SMC subunits (RAD21 and SCC3)..
• Shapes the genome in interphase by forming chromatin loops,.and holds together the sister chromatids following DNA replication until anaphase onset.
• Structure: ring-like that encircles to links sister chromatids together.
• Cohesin forms a ring-like structure that relies on other proteins and rings to provide genome stability.