Questions and Answers
What type of replication is DNA synthesis?
Bidirectional and semi-conservative
What are the roles of helicase and gyrase in DNA replication?
Helicase unwinds DNA, and gyrase prevents supercoiling.
What are Okazaki fragments?
Short segments of DNA on the lagging strand.
Which of the following eukaryotic DNA polymerases is primarily involved in mitochondrial DNA synthesis?
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What is the function of telomerase?
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Which type of repair is specifically distinct for removing bulky DNA damage?
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What is the primary role of DNA ligase in DNA replication?
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Why do RNA polymerases require a primer for synthesis?
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DNA synthesis occurs only in the 3' to 5' direction.
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Which of the following is a type of base involved in the structure of DNA?
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What are histones?
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Eukaryotic DNA has a single origin of replication per chromosome.
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What is the principle behind the structure of double-stranded DNA?
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How do the major and minor grooves in DNA affect protein binding?
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What binds to the outside of the nucleosome to assemble chromatin into a 30nm solenoid structure?
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What are the three major classes of RNA?
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What is the function of RNA polymerase?
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What is the role of HAT?
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What is an inhibitor of RNA polymerase II?
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What happens during splicing of hnRNA?
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The 5'-Cap structure on mRNA is added to the _____ end.
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What is the primary structure involved in ribosome function?
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What modifications occur to eukaryotic mRNAs?
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What is the function of tRNA?
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Prokaryotic mRNA is monocistronic.
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Alternative splicing allows for different protein isoforms from the same gene.
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What are SNARE proteins involved in?
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What functions does the endoplasmic reticulum serve?
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NSF (N-ethylmaleimide-sensitive factor) is required for ______.
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What is the role of SNAP in relation to NSF?
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Which Rab GTPase is required for early endosome fusion?
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The acidic lumen of lysosomes contains over 60 hydrolytic enzymes.
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What modifications are required for the targeting of lysosomal proteins?
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What leads to Tay-Sachs disease?
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What is the main role of autophagy?
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Alzheimer’s disease involves the formation of neurofibrillary tangles and senile plaques.
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What type of collagen is abundant in the body?
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What is required for the hydroxylation of proline during collagen synthesis?
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Match the following proteins with their functions:
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What is Marfan syndrome caused by?
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What do matrix metalloproteinases (MMPs) do?
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Study Notes
DNA Replication
- DNA replication is bidirectional, semi-conservative, and requires various proteins like helicase and gyrase.
- Helicases unwind the DNA, while topoisomerases, such as DNA gyrase, prevent supercoiling.
- DNA polymerases synthesize DNA in the 5’-to-3’ direction and require a primer, typically an RNA oligonucleotide created by primase.
- At the replication fork, synthesis occurs continuously on the leading strand and discontinuously on the lagging strand via Okazaki fragments.
- Eukaryotic DNA polymerases include α (initiation), β (repair), γ (mitochondrial), δ (lagging strand), and ε (leading strand).
- Telomerase extends telomeres, adding repetitive sequences to chromosome ends, counteracting the shortening that occurs during replication.
- Reverse transcriptase synthesizes DNA from an RNA template, utilized by retroviruses like HIV.
DNA Repair Mechanisms
- Mutagens can alter DNA structure, leading to mutations or cancer.
- DNA repair includes nucleotide excision repair, which removes bulky lesions, and base excision repair, for smaller base alterations.
- Mismatch repair detects and fixes base-pair mismatches during DNA replication using parental DNA as a template.
- Transcription-coupled repair focuses on actively transcribed genes but remains less understood.
- Human DNA repair defects, such as xeroderma pigmentosum and ataxia-telangiectasia, lead to increased cancer risk and require special precautions.
Nucleic Acids & Chromatin Structure
- DNA consists of nucleotides with a nitrogenous base (A, G, C, T), a sugar (deoxyribose), and a phosphate group; RNA contains uracil instead of thymine and ribose as sugar.
- DNA is structured as a right-handed double helix with antiparallel strands; hydrogen bonding between bases defines specific pairing (A=T, G≡C).
- B-DNA is the most stable and abundant form of DNA, with major and minor grooves that provide binding sites for proteins.
- Chromatin structure includes histones, which package DNA into nucleosomes, and nonhistone proteins providing additional structure and regulation.
- Acetylation of histones alters their interaction with DNA, facilitating transcription in euchromatin regions.
Transcription and RNA Synthesis
- Transcription generates mRNA, tRNA, and rRNA for protein synthesis, occurring in the nucleus for eukaryotes.
- Promoter sequences are critical for the binding of RNA polymerases, which initiate transcription.
- Eukaryotic RNA polymerases (e.g., RNA polymerase II) synthesize mRNA and are inhibited by certain agents.
- Eukaryotic mRNA undergoes capping, polyadenylation, and splicing, involving intron removal and exon joining.
- Alternative splicing allows for variations in mRNA production, exemplified by immunoglobulin light chain mRNA.
- Prokaryotic mRNA transcripts lack introns and are typically polycistronic, unlike eukaryotic mRNA.### Basic RNA Types
- Three major classes of RNA: tRNA (transfer RNA), rRNA (ribosomal RNA), mRNA (messenger RNA).
- mRNA carries DNA-encoded information to cytoplasm for protein synthesis.
- tRNA acts as carriers for amino acids during translation.
- rRNA is a main component of ribosomes essential for protein synthesis.
- All RNA types are synthesized by RNA polymerases in the nucleus, except a small percentage produced in mitochondria.
- RNA processing is necessary for the maturation of all RNA types, occurring within the nucleus.
Gene Transcription
- Bacterial genome has about 3000 genes; humans have roughly 25,000 genes.
- In specific human tissues, around 10,000 genes may be transcribed at any time; some are constitutive, while others are induced.
- Steps of transcription: promoter search, RNA synthesis, termination, post-transcriptional modifications.
- Key difference between prokaryotic and eukaryotic transcription processes.
Promoter and RNA Polymerase
- Promoter sequences are essential for initiating transcription, varying between prokaryotes and eukaryotes.
- RNA polymerase unwinds DNA and starts transcription downstream of the promoter.
- Eukaryotic RNA polymerase is a large enzyme composed of multiple subunits (10 protein subunits, 500,000 daltons).
- Most eukaryotic genes contain enhancer sequences not present in prokaryotes.
RNA Polymerases in Eukaryotes
- Three major types of RNA polymerases:
- RNA polymerase I: transcribes rRNA in the nucleolus.
- RNA polymerase II: transcribes hnRNA, the precursor to mRNA, in the nucleoplasm.
- RNA polymerase III: transcribes small RNAs (e.g., tRNAs, snRNAs).
mRNA Processing Modifications
- Three main modifications to eukaryotic mRNA:
- Capping: Adds a 5' cap (methylguanosine) to stabilize the mRNA and assist in translation.
- Polyadenylation: Addition of a poly-A tail for stability; the tail shortens over time due to degradation.
- Splicing: Introns are removed, and exons are joined by spliceosomes; small nuclear RNAs (U1-U6) are involved in this process.
- Alternative splicing can generate different mRNA variants from a single gene.
Differences between Prokaryotic and Eukaryotic mRNA
- Prokaryotic mRNA: polycistronic, short-lived, translated immediately.
- Eukaryotic mRNA: monocistronic, contains introns and exons, long-lived, must undergo processing.
Translation (Protein Synthesis)
- Participants in protein biosynthesis: mRNA, tRNA, ribosomes, GTP, and various factors.
- Process: mRNA is translated from 5' to 3'; proteins synthesized from amino to carboxy terminal.
- Codons consist of three nucleotides, each coding for one amino acid.
- Ribosomes have P (peptidyl) and A (acceptor) sites.
tRNA Structure and Function
- tRNAs are approximately 80 nucleotides long, cloverleaf-shaped, with an L-shaped 3D structure.
- Amino acids are attached to the 3' end; codon-anticodon pairing ensures correct amino acid incorporation.
Ribosome Structure
- Ribosomes composed of large and small subunits that contain rRNA and proteins, critical for protein synthesis.
Aminoacyl tRNA Synthetases
- 20 distinct synthetases, each specific to an amino acid, are needed to attach amino acids to tRNAs.
- Activation of amino acids requires ATP, converting it to AMP.
Translation Process
- Initiation requires initiation factors, mRNA, and methionine-tRNA binding to small ribosomal subunit.
- Elongation involves GTP-driven delivery of tRNA, peptide bond formation, and ribosome translocation.
- Termination occurs at stop codons, leading to peptide release and ribosomal disassembly.
Secreted and Membrane-Resident Proteins
- Secreted proteins have signal peptides directing them to the endoplasmic reticulum (ER) for translation.
- Membrane proteins possess stop transfer sequences aiding in anchoring within the membrane.
Cellular Organelles Overview
- Endoplasmic Reticulum (ER):
- Structure: Network of tubules and sheets; maintained by proteins and microtubule motor proteins (kinesins and dyneins).
- Functions: Synthesis of secreted and membrane proteins, calcium storage, lipid biosynthesis.
- Golgi Complex: Series of flattened sacs for protein modification, with cis, medial, and trans compartments.
Endocytosis
- Process of nutrient uptake, signaling, and pathogen entry involves clathrin-coated vesicles and requires proteins like dynamin and AP2.
Lysosomes
- Contain hydrolytic enzymes essential for breakdown of cellular components.
- Dysfunction leads to lysosomal storage diseases (e.g., Tay-Sachs disease) causing severe health issues.
Autophagy
- Involves formation of autophagosomes for degrading cellular components, regulated by signals such as nutrient availability.
- Stages include initiation, nucleation, expansion, closure, and lysosomal fusion.### Autophagy in Disease
- Autophagy is implicated in various diseases, with a focus on Alzheimer's disease (AD).
- Alzheimer's disease, the most prevalent form of senile dementia, features senile plaques (β-amyloid) and neurofibrillary tangles (Tau protein).
- The exact role of β-amyloid in AD pathogenesis remains uncertain; it results from proteolytic cleavage of amyloid precursor protein (APP).
- Impaired autophagy in AD leads to an accumulation of immature autophagosomes.
Extracellular Matrix (ECM) Components
- The extracellular matrix comprises three major classes of proteins: structural proteins, proteoglycans, and specialized adhesion proteins.
- Structural proteins include collagen, elastin, and fibrillin, providing structural integrity.
- Proteoglycans are essential for forming a gel-like ECM, while adhesion proteins like fibronectin and laminin facilitate cell attachment.
Collagen
- Collagen is a diverse family of fibrous proteins, with 28 known types, primarily produced by fibroblasts.
- Type I collagen is the most abundant, composed of 33% glycine, 21% proline, and hydroxyproline.
- Collagen exhibits a helical structure with repeating sequences designated as (Gly-X-Y), and most types have a triple helical formation.
- Fibrillar collagens (Types I, II, III) account for 90% of total collagen; non-fibrillar types include IV, VIII, and X.
Collagen Synthesis and Modifications
- Synthesized as a preproprotein, collagen undergoes enzymatic processing, involving the removal of N and C-terminal extensions.
- Post-translational modifications include the hydroxylation of proline and lysine residues, dependent on vitamin C, essential for collagen stability.
- Crosslinking of collagen fibrils increases tensile strength, vital for its structural role.
Clinical Relevance of Collagen
- Scurvy is caused by vitamin C deficiency, leading to impaired collagen hydroxylation, resulting in symptoms like bleeding gums and poor wound healing.
- Osteogenesis imperfecta, a genetic disorder, results in faulty collagen production and is treatable with bisphosphonates to inhibit osteoclasts.
Elastin
- Elastin is a high molecular weight protein that imparts elasticity, formed by crosslinked lysines.
- This stable molecule has a long half-life, supporting tissue elasticity in various organs.
Fibrillin
- Fibrillin is a large glycoprotein essential for microfibril structure in connective tissues.
- Marfan syndrome results from mutations in the fibrillin gene, presenting with tall stature and arachnodactyly.
Proteoglycans
- Proteoglycans consist of core proteins covalently linked to glycosaminoglycan chains, crucial for ECM viscosity.
- Found in synovial fluid, vitreous humor, arterial walls, and bone, they consist of seven major glycosaminoglycans.
Synthesis and Degradation of Proteoglycans
- Proteoglycan synthesis begins in the ER with sugar attachment to serine residues, utilizing UDP-sugars.
- Sulfation occurs post-sugar addition, essential for their functionality.
- Mucopolysaccharidoses are disorders resulting from proteoglycan degradation issues.
Integrins
- Integrins are adhesion receptors that anchor cells to the ECM, with 24 known types that enable cell migration and adhesion.
Adhesion Proteins: Fibronectin and Laminin
- Fibronectin is a glycoprotein consisting of two peptide chains, facilitating various cellular functions and ECM interactions.
- Laminin, composed of three polypeptide chains, plays a critical role in forming basal laminae and binding to type IV collagen.
Matrix Metalloproteinases (MMPs)
- MMPs are a group of over 20 proteolytic enzymes responsible for degrading ECM proteins, crucial for matrix turnover and tissue remodeling.
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Test your knowledge on DNA replication and repair processes with this quiz. Focus on key factors involved like helicase, gyrase, and the role of DNA polymerase. Understand the bidirectional and semi-conservative nature of DNA synthesis along with the implications of Okazaki fragments.
