Genetics Final Exam PDF

GENETICS FINAL EXAM CHAPTER 7: DNA STRUCTURE AND REPLICATION 4 Criteria of DNA 1. Information: necessary information to make an organism 1. Transmission: passed from parent to offspring 2. Replication: make copies of itself for inheritance 3. Variation...

GENETICS FINAL EXAM CHAPTER 7: DNA STRUCTURE AND REPLICATION 4 Criteria of DNA 1. Information: necessary information to make an organism 1. Transmission: passed from parent to offspring 2. Replication: make copies of itself for inheritance 3. Variation: explain phenotypic diversity Central Dogma DNA → RNA → Protein Transcription: DNA → RNA Translation: RNA → Protein Epigenetics: DNA can be chemically modified and inherited. RNA Types that regulate and don’t lead to protein: miRNA, siRNA, lncRNA, piRNA Key Experiments Leading to DNA as Genetic Material 1. Frederick Griffith (1928) - Transformation in Bacteria ○ Studied Streptococcus pneumoniae ○ Strains: Type S (smooth): Lethal, protective capsule. Type R (rough): Non-lethal, no capsule. ○ R strain became virulent(S) when mixed with heat-killed strain ○ transformation: Something from the dead S cells transformed R cells into virulent S cells 2. Avery, MacLeod, and McCarty (1944)--Found DNA was a transforming agent ○ Only destroying DNA of type S cells prevent live S strain from recovering 3. Hershey and Chase (1952) - Found DNA entered the E. coli cells, confirming DNA as the genetic material. ○ Studied the bacteriophage T2 virus (only has DNA and protein). ○ Isotope Labeling: 32P labels DNA. 35S labels protein. ○ Used radioactive phages to infect E. coli, then separated the bacteria from phage ghosts. The Structure of DNA (1953) Watson & Crick: Created 1st correct DNA model using Rosalind Franklin's X-ray diffraction data DNA Structure: ○ Double helix: Two strands coiled around each other. ○ Nucleotides: building blocks ○ Components of Nucleotides: 1. Phosphate group 2. Deoxyribose sugar 3. Nitrogenous base (A, T, C, G) DNA Nucleotide Structures Phosphomonoester bond: ribose sugar to phosphate  Glycosidic bond: ribose sugar to base Purines (Adenine & Guanine): ○ Two-ring structure. ○ Larger ○ more prone to mutation and require repair mechanisms. Pyrimidines (Cytosine & Thymine): ○ Single-ring structure. ○ Cytosine is often methylated(5’) in the genome, particularly when followed by guanine. Chargaff’s Rules of Base Pairing Rule: [A] = [T] and [C] = [G] [T + C] = [A + G] A+T≠G+C helped confirm that DNA is double-stranded and complementary in structure. Pyrimidine + Pyrimidine: Too thin Purine + Purine: Too thick Purine + Pyrimidine: Ideal thickness (fits X-ray crystallography), supporting double-helix The Structure of DNA Anti-parallel Strands:One strand runs 5’ to 3’, the other runs 3’ to 5’. Base Pairing: ○ (A) pairs with (T): 2 hydrogen bonds. ○ (C) pairs with (G): 3 hydrogen bonds Hydrophobic Base Stacking: ○ Bases stack flat on top of each other, excluding water (hydrophobic interactions). ○ Hydrophilic sugar-phosphate backbone interacts with water and proteins. Backbone: ○ Phosphodiester bond links nucleotides in a strand. ○ negatively charged backbone (phosphate groups) neutralized by + charged proteins DNA Double Helix Structure Two Strands: ○ Coiled in a right-handed direction. ○ 10 base pairs per complete twist. Grooves: ○ Major Groove: Most proteins (e.g., transcription factors) bind here. ○ Minor Groove: Smaller, TATA box binding, Distorts DNA structure to open up major groove Helix Stabilization: ○ Hydrogen bonds between complementary bases. ○ Base stacking interactions (Van der Waals forces) Models of DNA Replication 1. Semiconservative Replication: Each parent strand serves as a template for a new strand  2. Conservative Replication: Parent strands remain intact, and new strands are synthesized. 3. Dispersive Replication: Parental and new DNA segments are mixed in along the chromosomes. Experimental Evidence for Semiconservative Replication Meselson & Stahl (1958): Cesium chloride centrifugation: Used heavy nitrogen (N15) and light nitrogen (N14) to label DNA. Experiment: E. coli grown with 15N, then switched to 14N and DNA was separated by density. 1st Gen: Intermediate density (one heavy and one light strand). 2nd Gen: A mix of light and intermediate density. Semiconservative DNA Replication—Arthur Kornberg Deoxyribonucleotides (dATP, dCTP, dGTP, dTTP) in triphosphate form. DNA Polymerase (enzyme) to synthesize DNA. Kornberg purified DNA polymerase I. Kornberg’s Experiment: DNA replication in test tubes Used wrong polymerase, but still synthesized small DNA pieces. DNA template separated without helicase. DNA polymerase I has limitations: too slow and dissociates after 20-50 nucleotides Semiconservative Replication Mechanism Helicase: Unwinds the DNA. DNA Polymerase III: Synthesizes the new strand in 5’ to 3’ direction. Primase: Adds short RNA primers to initiate DNA synthesis. Ligase: Joins Okazaki fragments on the lagging strand. DNA Replication Speed & Accuracy Speed: In E. coli, DNA polymerase III synthesizes 2,000 nucleotides per second. Accuracy: 1 error per 1010 nucleotides. Proofreading: ○ DNA polymerase III: Has 3’ to 5’ exonuclease activity to correct errors. ○ If polymerase III misses an error, DNA polymerase I fixes it. DNA Polymerases in E. coli 1. DNA Polymerase I (PolA):Major repair enzyme. ○ 3’ to 5’ exonuclease: Removes mismatched bases. ○ 5’ to 3’ exonuclease: Degrades double-stranded DNA. 2. DNA Polymerase II (PolB):Replication restarts after damage. 3. DNA Polymerase III (PolC):Main enzyme for replication, synthesizes long DNA strands. ○ 3’ to 5’ exonuclease activity: Corrects mismatches during synthesis. 4. DNA Polymerase IV (DinB) and V (UmuD’2C):Involved in error-prone repair mechanisms. Replicating the Bacterial Chromosome: John Cairns incorporated 3H-thymidine into E. coli DNA and visualized it w/ photographic emulsion. He studied DNA synthesis on the E. coli chromosome:  ○ After one round of replication: The chromosome is a double-stranded circular DNA molecule. ○ 2nd round: DNA polymerase synthesizes DNA in two directions by both strands simultaneously Key Concepts of DNA Replication DNA Replication Fork: Site where DNA is unwound and replication occurs. Template: RNA primer provides 3' OH for DNA polymerase to start replication. Antiparallel Strands: ○ Lagging Strand: Synthesized in short pieces (Okazaki fragments) moving away from fork (5’ -3’). ○ Leading Strand: Synthesized continuously towards the fork (5’ to 3’). Steps in DNA Replication(synthesis of lagging strand) 1. Primer synthesis: primase synthesizes RNA primer(5’-3’) to provide 3’ OH group for DNA polymerase 2. Leading strand: DNA polymerase III elongates the leading strand continuously towards the replication fork 3. Lagging strand: DNA synthesized in okazaki fragments, each starting with an RNA primer 4. RNA removal: DNA polymerase I cleaves RNA primers, fills in gaps, and synthesizes DNA(5’-3’ direction) 5. Ligation: DNA ligase seals the gap between okazaki fragments by forming phosphodiester bonds Key Enzymes and Proteins in DNA Replication protein/enzyme function Helicase unwinds DNA double helix ahead of replication fork DNA gyrase(topoisomerase) Relieves supercoiling tension caused by unwinding DNA Primase Synthesizes RNA primers(providing 3’ OH) for both strands DNA polymerase III Extends the RNA primers with new DNA(leading strand and okazaki fragments) DNA polymerase I Removes RNA primers, replaces with DNA, and proofreads(3’-5’ exonuclease) DNA ligase Seals gaps between okazaki fragments, forming a continuous DNA strand Single-strand binding protein(SSB) Bind single-stranded DNA to prevent rejoining and degradation Replication Bubble in Prokaryotes (E. coli) OriC: The origin of replication in E. coli (circular genome). Replication occurs in both directions from the origin, forming two replication forks. Primosome: A complex of primase and helicase that leads the replication fork. Replisome: Multi-protein complex that carries out DNA synthesis. DNA Replication in Eukaryotes Multiple Origins of Replication Occurs during S Phase DNA wrapped around histones and needs to be unwound for replication. Telomerase maintain chromosome ends to prevent shortening  Telomeres & Telomerase Telomeres: protective repeated DNA sequences(TTAGGG) at ends of chromosomes Telomerase: Helps maintain telomere length ○ RNA part: Serves as a template for extending 3' overhang of telomeres by adding DNA sequence Problems at Chromosome Ends (Telomere Shortening) Lagging Strand: After last RNA primer is removed, a gap remains at 3' end of lagging strand. Telomere Shortening: Each round of replication results in loss of a small portion of the telomere. Telomeric Cap Structure (T-loop) ○ T-loop: A protective loop formed by 3' overhang of telomeres to stabilize chromosomes ○ Telomere Binding Proteins: TRF1 and TRF2 stabilize telomere and protect chromosome ends from being recognized as DNA breaks. Elongation Process: Polymerase activity of telomerase extends the telomere. RNA moves along DNA, adding repeats to the 3' end. Primase adds an RNA primer at the end. DNA polymerase fills in the gap created by the primer. DNA ligase seals the final gap. Werner Syndrome(WRN): defective telomere maintenance, leading to premature aging Cancer Cells: ↑ telomerase activity, allowing them to divide infinitely and avoid cellular senescence. Telomeres/Telomerase protecting ends discovery: Elizabeth Blackburn, Carol Greider, and Jack Szostak Feature bacteria(prokaryotes) eukaryotes RNA synthesis and translation - occur simultaneously - RNA synthesis occurs in the nucleus - translation occurs in the cytoplasm Location of transcription - cytoplasm - nucleus Translation timing - ribosomes begin translating mRNA before - translation occurs after RNA processing and transcription finishes transport to the cytoplasm Cell complexity - less complex - more complex RNA processing - no processing required - 5’ capping, splicing, and 3’ polyadenylation Ribosome location - ribosomes are free in the cytoplasm - ribosomes free in the cytoplasm or attached to ER RNA vs DNA Feature RNA DNA Sugar Ribose(2’ OH) Deoxyribose(2’ H) Stability Less stable(single-stranded) More stable(double-stranded) Bases (U) replaces (T) Thymine(T) Methyl group replaced by hydrogen Methyl group at 5’ position Function Single-stranded Double helix structure can form secondary structures(hairpins) Makes it unstable(broken chain) Types of RNA Type Function mRNA Intermediary transferring genetic info from DNA to ribosomes for protein synthesis tRNA Transfers AAs to ribosomes for protein assembly rRNA Structural and catalytic components of ribosomes Most abundant RNA* snRNA Small nuclear RNAs involved in splicing and RNA processing miRNA Regulate mRNA stability and translation, linked to cancer siRNA Small interfering RNAs, involved in RNA interference piRNA Piwi-interacting RNAs, protect genome integrity lncRNA Long non-coding RNAs, involved in epigenetic regulation Ribozymes Catalytic RNAs capable of enzymatic activity CRISPR RNAs Guide RNA for genome editing Ribosomal RNA (rRNA) in Eukaryotic Cells: High quantity of rRNA needed for ribosome production, essential for protein synthesis Embryogenesis: High rRNA levels crucial for translation during early development. B4 fertilization, mom supplies ↑ RNA to egg, so pre-made ribosomes are available right after fertilization. RNA Synthesis Initiation 5' Flanking Region: region before the coding sequence, important for transcription initiation. Consensus Sequences: ○ AUG: The start codon for protein synthesis (first amino acid). ○ -35 and -10 Sequences: Found in most promoters. TTGACAT (−35) and TATAAT (−10) recognized by RNA polymerase. The distance between these sequences is 15-17 base pairs, forming the TATA box. RNA Synthesis and Transcription  1. Initiation: ○ Prokaryotes: core enzymes (α2, β, β', ω subunits) and a sigma factor (σ). tΣ70 directs RNA pol. to promoter region at -10 and -35, starting transcription at +1 site. After initiation, the sigma factor dissociates, and RNA polymerase continues elongation. ○ Eukaryotes: Requires multiple transcription factors and promoter regions (e.g., TATA box). 2. Elongation: ○ RNA polymerase unwinds DNA and synthesizes RNA in the 5′ to 3′ direction. ○ Nucleotides added to 3′ OH of growing RNA chain through nucleophilic attack by 5’ phosphate 3. Termination: ○ Terminator sequences (around 40 bp), rich in GC followed by A's, form hairpin structure in RNA. ○ hairpin disrupts RNA-DNA so RNA polymerase pause, backtrack, and detach from DNA template ○ The U-A hybrid is weak, leading to the release of RNA and RNA polymerase. ○ 2nd mechanism: Rho-dependent: Rho factor binds RNA and unwinds DNA-RNA, dissociating RNA Eukaryotic RNA Processing 1. 5′ Capping: Adds modified guanine nucleotide (5′-5′ triphosphate linkage) to protect RNA. 2. Splicing: Removes introns (non-coding sequences) to produce mature mRNA. 3. 3′ Polyadenylation: Poly(A) tail added by poly(A) polymerase to protect RNA and aid in nuclear export. mRNA Decay(E.coli) 1. Pyrophosphohydrolase: Initial pyrophosphate removal 2. Internal cleavage: RNase E cleaves mRNA internally 3. 3’ exonucleolytic decay: exonuclease chews mRNA from 3’ end to degrade fully Miller Spread Technique: Visualize transcription under an electron microscope Multiple RNA molecules transcribed simultaneously from a single DNA template Prokaryotic vs. Eukaryotic Transcription Prokaryotes Eukaryotes Location Cytoplasm Nucleus mRNA processing No processing(mRNA naked) 5’ capping, polyadenylation, splincing mRNA export Immediately available for translation Must pass through nuclear pore to cytoplasm Translation simultaneously w/ transcription in cytoplasm Occurs in cytoplasm, after mRNA processing ribosomes Free in cytoplasm Free in cytoplasm or bound to ER Protein synthesis Immediately after transcription After mRNA export and processing Transcription Sigma factor guides RNA polymerase Promoters, enhancers, transcription factors regulation 1957 Volker & Astrachan: Showed RNA was the information transfer intermediate b/ween DNA and protein Showed RNA synthesis and rapid turnover using radioactive uridine in a pulse-chase experiment. RNA Polymerases in Eukaryotes RNA Polymerase Location Function Involved in RNA Pol I nucleolus - Transcribed rRNA genes(except 5S) Ribosome synthesis RNA Pol II nucleus - Transcribes mRNA, long non-coding RNA, Transcription of most genes for microRNA, some snRNA protein synthesis RNA Pol III nucleus - transcribes small RNA genes Protein synthesis(translation) RNA Polymerase II Transcribed Genes Promoter-proximal elements: upstream activator sequences(UAS) near promoter Control elements:Bind transcription factors for gene-specific expression (ex. muscle-specific factors) ○ (~100 bp upstream) Enhancer elements: located upstream or downstream, ~1000 bp away TATA Box: DNA unwinding point. Consensus Sequence: TATANT (found in ~50% of genes). Recognized by TATA Binding Protein (TBP) as part of the TFIID complex. Binds DNA's minor groove using a β-sheet and distorts DNA structure to recruit other transcription factors. Saddle structure Transcription Initiation 1. Pre-Initiation Complex (PIC): 6 General Transcription Factors (GTFs) and RNA Polymerase II ○ TBP binds the TATA box, and GTFs guide RNA Polymerase II to the transcription start site. 2. TFIIH: causes RNA Polymerase II to start transcription and dissociates from most GTFs. ○ Helicase: Unwinds DNA. ○ Kinase: Phosphorylates RNA Polymerase II's C-terminal domain (CTD). Transcription Elongation CTD Phosphorylation: binding sites for other proteins involved in processing ○ Serine at positions 2 and 5 of YSPTSPS repeat is phosphorylated (26 in yeast, 52 in humans). ○ Phosphorylation recruits enzymes for RNA processing. RNA Processing During Elongation: ○ 5’ Capping: Protects RNA from degradation, serves as a translation initiation site Involves triphosphatase (TP), guanylyltransferase (GT), and methyltransferase. Structure: 7-methylguanosine linked 5’-5’.  Viruses use enzymes this to stop translation and take over cell by destroy 5’ cap proteins 1. Triphosphatase: takes away phosphate group from first nucleotide 2. Guanylytransferase: adds a new piece of guanosine monophosphate 3. Methyltransferase: attaches methyl group to RNA strand to further stabilize cap ○ Splicing: Removes introns and joins exons with help of small nuclear RNAs (snRNAs). ○ Polyadenylation: Adds A residues to 3’ end, protected by Poly-A binding proteins. CPSF (Cleavage and Polyadenylation Specificity Factor) binds AAUAAA sequence. Cleavage by Poly-A Polymerase (PAP), followed by adenine addition. Protect 3’ end and fold over to interact with proteins bound to cap structure Leaves complex behind sitting on TATA box waiting for other transcription factors to recycle off Transcription Pausing: Allows rapid response to stress (e.g., heavy metals, heat)(~50 bp) Negative Factors: NELF (Negative Elongation Factor) and DSIF (DRB Sensitivity-Inducing Factor). Positive Factor: P-TEF-b (Positive Transcription Elongation Factor B). Balance b/ween (-) trying to pause and (+) that tells it to phosphorylate (-) protein Mechanism: P-TEF-b phosphorylates RNA Polymerase II (CTD), resuming elongation. Transcription Termination 1. Cleavage and Polyadenylation:Signals RNA cleavage and poly-A tail addition. 2. Termination Models: ○ Torpedo Model:Exonuclease Xrn2 degrades RNA, releasing RNA Polymerase II. ○ Allosteric Model:Conformational changes in RNA Polymerase II cause dissociation. Summary Table: Eukaryotic Transcription Stage Initiation - TATA box recruits TBP and GTFs to form the PIC - TFIIH phosphorylates RNA polymerase II for activation Elongation - RNA polymerase II phosphorylated CTD recruits processing enzymes Pausing - regulated by NELF, DSIF, and P-TEF-b to enable quick stress response Processing - co-transcriptional modifications: 5’ cap, splicing(intron removal), and 3’ poly A tail Termination - torpedo(Xrn2 exonuclease) or allosteric(conformational change) models release RNA pol II Richard Roberts and Philip Sharp—Eukaryotic genes are discontinuous, containing exons and introns. Duchenne Muscular Dystrophy (DMD) Gene—vast majority of bps are removed as introns RNA in eukaryotes is made as precursors Purpose of Introns: ○ Provide nucleotide triphosphates (via degradation). ○ regulatory roles (e.g., siRNAs).  ○ Fold up on themselves to create double stranded RNA after removed ○ Cleaved to becomes silencing RNAs Conserved Sequences in Intron Splicing Region Conserved Sequence Location 5’ Splice Donor GU Exon-intron boundary(5’) 3’ Splice Acceptor AG Intron-exon boundary (3’) Branch Point A 15-45 bp upstream of 3’ splice site Riboprotein Complex Spliceosome: splicing nuclear mRNAs RNA-induced silencing complex(RISC): mRNA silencing Ribosome: translation Accidental discovery through research on autoimmune diseases like systemic lupus erythematosus Autoimmune disease: mistakenly produce antibodies against spliceosome, treating it as foreign substance Splicing Mechanism Two-Step Transesterification: ○ Step 1: 2’-OH of branch point A attacks the 5’ splice site phosphate. Forms a 2’-5’ phosphodiester bond → intron lariat loop. ○ Step 2:3’-OH of exon 1 attacks the phosphate of exon 2. Exons join; lariat intron is released. Spliceosome Assembly Components: ○ 5 snRNAs: U1, U2, U4, U5, U6. ○ snRNPs and >100 proteins. ○ Interaction with CTD (C-terminal domain of RNA Pol II). Steps: ○ U1 binds 5’ splice donor; U2 binds branch point A. ○ RNA complementarity ensures precise binding. ○ Protein components fold RNA to bring exon boundaries together. Catalyzes removal of intron through reactive hydroxyls Self-Splicing Introns—Tom Cech (1989 Nobel Prize) Introns self-splice without protein assistance. RNA acts as a ribozyme. Requires a guanosine cofactor for transesterification by providing reactive OH Folding of intro forms full circle before destroyed and brings reactants together  Make guide RNA into virus and change complementarity, you can take out viral RNA sequence & destroy it Alternative Splicing: Produces multiple proteins from one gene by including/excluding different exons. Examples: 1. Rat α-tropomyosin: Tissue-specific exon selection. 2. FGFR2: Alternative isoforms of fibroblast growth factor receptors bind different ligands. Four Types: 1. Exon skipping (most common). May be protein on exon-intron boundary and blocks joining 2. Alternative 3’ splice sites. 3. Alternative 5’ splice sites. 4. Mutually exclusive exons. DMD: altered splicing due to deletions and mutations in dystrophin gene mRNA Decay—Key Enzymes Deadenylase: Removes poly-A tail. Decapping Enzyme: Removes 5’ cap. Exonucleases degrade mRNA from 3’-5’ or 5’-3’. miRNA and siRNA: Gene Regulation Feature miRNA siRNA Mechanism Translation inhibitor RNA cleavage Complementarity with Target Imperfect Perfect Function Regulates many genes Targets specific mRNAs (~1900 miRNA in humans) Processed from Processed from hairpin RNA by dicer Processed from long dsRNA miRNA Translation Inhibition Mechanisms 1. miRNA transcribed and folds into a double-stranded structure due to complementarity. 2. Dicer Cleavage: recognizes double-stranded RNA and cleaves it into ~22-nucleotide fragments. 3. RISC Assembly: RISC uses its helicase activity to separate the two strands of the miRNA fragment. One strand becomes biologically active single-stranded miRNA. 4. active miRNA guides RISC to a target by binding to 3’ untranslated region (3’UTR) of the mRNA. 5. Action on Target mRNA: Degradation: RISC can degrade target mRNA if complementarity is high. Translation Repression: If complementarity is partial, translation of mRNA is repressed Outcome: target gene’s expression in inhibited, by mRNA degradation or inhibiting its translation Ex. lin-4 represses lin-14 and lin-28 in C.elegans Ex. mRNA regulate programmed cell death(apoptosis) to take out regions between digits siRNA Pathway  1. Dicer: Processes dsRNA into ~21-22 nucleotide siRNA fragments. 2. RISC Complex: ○ Helicase activity creates single-stranded RNA. ○ Guide strand binds perfectly to target mRNA. ○ Argonaute (AGO) cleaves target mRNA. 3. Outcome: Cleaved mRNA is degraded by exonucleases. RISC Models for Repression of miRNA Translation: 1. Interference with Translation Initiation - Cap Binding Block: RISC prevents binding of translation initiation factors to 5’ cap structure so mRNA translation ↓ 2. Interference with Translation Initiation - Ribosome Assembly Block: RISC blocks ribosome assembly at 5’ end of the mRNA. Prevents the ribosome from attaching and initiating protein synthesis. 3. Interference with Elongation: binds mRNA, causing physical block that halts elongation of polypeptide chain during translation. 4. Removal of the Poly-A Tail: RISC promotes deadenylation, leading to the removal of the poly-A tail. Tail loss disrupts interaction b/ween poly-A binding proteins & cap structure, destabilizing mRNA Three Experiments Demonstrating Gene Silencing 1. Fire and Mello (2004 Nobel Prize) – Injection of dsRNA ○ Process: Double-stranded RNA (dsRNA) of the unc-22 gene injected into C. elegans embryos ○ Result: Adult worms displayed muscle defects, indicating silenced unc-22 gene ○ Conclusion: Muscle-specific unc-22 gene shut down by dsRNA.. 2. Jorgensen – Insertion of Transgene ○ Process: A transgene (produce pigment) was inserted into petunia cells that grow into plants ○ Result: Flowers had white sectors, indicating a loss of pigmentation. ○ Conclusion: transgene and the plant's endogenous pigment gene were silenced. due to dsRNA produced by transgene, leading to "cosuppression." 3. Baulcombe – Insertion of Viral Gene ○ Process: viral gene was inserted into tobacco plants and plants were then exposed to virus ○ Result: Plants remained healthy and showed no symptoms of viral infection. ○ Conclusion: The viral gene was transcribed into dsRNA, preventing virus replication Antibiotics and Ribosomes antibiotics target bacterial ribosomes while eukaryotic ones stay untouched. Antibiotic resistance: Mutations in bacterial rRNA or ribosomal proteins Peptide Bond Formation Amino acids (AAs): amino group (NH₂) and carboxyl group (COOH). ○ Example AAs: cysteine (contain sulfur) Serine, threonine (contain hydroxyl groups) Covalent bond: peptide bond b/ween amino end &carboxyl end of another, releasing 1 molecule of water. Levels of Protein Structure Primary structure: Sequence of AAs (linear). Secondary structure: ○ Alpha helix: 3.6 AAs per turn of the helix. ○ Beta-pleated sheet: AAs running in opposite directions, stabilized by hydrogen bonds. Tertiary structure: disulfide bonds between cysteine residues. ○ Stabilized by various interactions, such as binding with heme groups. Quaternary structure: Multiple polypeptide chains join(e.g., heme with alpha and beta subunits). Cracking the Genetic Code DNA Structure (1953): linear nucleotide sequence in DNA corresponds to linear AA sequence in proteins. mRNA and Protein Sequence: mRNA (5’ to 3’) corresponds to protein (N-terminus to C-terminus). Overlapping vs. Non-overlapping Genetic Codes Overlapping code: single nucleotide can appear in multiple codons. Non-overlapping code: Each nucleotide is part of only one codon. Codon combinations: 4n Frameshift mutations: Insertion or deletion changes the reading frame. ○ Proflavin: chemical that can cause insertions or deletions rII Gene Mutation (Crick & Brenner) Proflavin mutation: mutation in rII gene of T4 phage causes it to grow only on one strain of E. coli. Suppressor mutation: 2nd mutation in the same gene can restore phage's ability to grow on both strains Cracking the Code: Marshall Nirenberg and Others (1961) 1. Synthetic RNA Experiment: Used poly U RNA with E. coli extract in vitro. ○ Produced polyphenylalanine peptide, identifying UUU as the codon for phenylalanine. 2. Repeating Sequence Test: Tested synthetic RNAs like (AGA)n. ○ Matched triplet codons to amino acids by analyzing resulting polypeptides. 3. Created a codon table mapping each triplet to its amino acid. 4. Radioactive Amino Acids: Tracked proteins synthesized from RNA using labeled amino acids. Genetic Code Characteristics Codons: 64 codons, 61 specify AAs, and 3 are stop codons (UGA, UAA, UAG). Start codon: Methionine (AUG) signals start of protein synthesis in prokaryotes and eukaryotes. Degeneracy of the code: Multiple codons can specify the same AA (e.g., 6 codons for arginine). Codon usage bias: Some organisms have preferences for certain codons. tRNA Structure and Specificity tRNA structure: Each tRNA is specific to an AA ○ AA attachment site on the 3’ end. ○ anticodon loop that recognizes the corresponding codon in mRNA.  ○ pseudouridine molecules: stabilize tRNA and are used in mRNA vaccines for increased durability. Alexander Rich's Contribution: Identified tRNA structure, linking design decoding mRNA into proteins. Wobble hypothesis: third nucleotide of the anticodon can pair with multiple bases in the codon Wobble Base-Pairing Rules: flexibility in base-pairing 5’ End of Anticodon 3’ end of codon A U G C or U U A or G I(Inosine) A, C, or U Ribosome Structure and Function Ribosome Component Prokaryotic Ribosome Eukaryotic Ribosome Small subunit 16S rRNA + 21 proteins(30S) 18S rRNA + 33 proteins(40S) Large subunit 23s, 5s, 5.85 rRNA + 33 proteins(50S) 28S, 5S, 5.85S rRNA + 49 proteins(60S) Total ribosome size 70S(30S +50S) 80S(40S +60S) rRNA functions: rRNA plays the major role in protein synthesis within the ribosome. ○ RNA polymerase I: Synthesizes 18S, 28S, and 5.8S rRNA. ○ RNA polymerase III: Synthesizes 5S rRNA. Ribosomal proteins are translated in cytoplasm, then imported into the nucleus to assemble with rRNA. Key Ribosomal Binding Sites A site: Binds the incoming aminoacyl-tRNA. P site: Holds the growing peptide chain. E site: Holds the tRNA ready to exit the ribosome. Protein Synthesis Overview—Key Components: Peptidyl Transferase: catalyzes peptide bonds, transferring growing polypeptide chain to the next AA ○ in the 50S ribosomal subunit Decoding Center: ensures only tRNAs with matching anticodons for the mRNA codon enter the A site. ○ in the 30S subunit tRNA: Transfers amino acids to the ribosome. ○ Deacylated tRNA (w/out an amino acid) is released from E site so ribosome moves to next codon Translation Factors:  Stage Bacteria Eukaryotes Function Initiation fMET-tRNAᶠᴹᵉᵗ Met-tRNAᵐᴱᵗ Initiator tRNA IF1 eIF1A Blocks A site IF2 eIF2, eIF5B Facilitates entry of initiator tRNA IF3 eIF3, eIF1 Blocks premature association of large ribosomal subunit eIF4 complex Unwinds mRNA, binds 5’ cap, and circularizes mRNA Elongation EF-Tu eEFIa Delivers aminoacyl-tRNA EF-G eEF2 Translocates ribosome Termination RF1, RF2 eRF1 Recognizes stop codons RF3 eRF3 Stimulates peptide release Protein Synthesis Stages 1. Initiation of Translation Prokaryotes (Bacteria) Shine-Dalgarno Sequence:pairs with 16S rRNA, positioning the first AUG codon in the P site in 5' UTR Initiation Factors (IF1, IF2, IF3): ○ IF1 & IF3: Prevent 50S subunit from associating with the 30S subunit prematurely. ○ IF2: Helps bring the initiator tRNA to the P site. ○ Formyl-methionine (fMet): first AA added to the initiator tRNA and placed in the P site. Eukaryotes Cap Binding Complex (eIF4A, eIF4B, eIF4G): Binds to the mRNA cap structure. Scanning Mechanism: complex moves along mRNA in the 5’ to 3’ direction, searching for the first AUG. Initiation Complex: When AUG is recognized, 60S ribosomal subunit joins 40S to form the 80S ribosome. 2. Elongation of the Polypeptide Chain 1. Aminoacyl-tRNA entry: tRNA carrying an amino acid enters the A site. 2. Peptide Bond Formation: amino acid in the A site forms a bond with the growing peptide in the P site. 3. Translocation: EF-G hydrolyzes GTP to GDP, shifting ribosome so that tRNAs move from A → P → E sites. 4. Exit: Deacylated tRNA leaves through the E site. 3. Termination of Translation Stop Codons: UGA, UAA, UAG. Release Factors (RF1, RF2, RF3): Recognize stop codons and release the polypeptide. Water Molecule: Enters peptidyl transferase center and triggers release of the polypeptide. Ribosomal Subunits: 30S & 50S subunits separate, allowing 30S subunit to form a new initiation complex. Posttranslational Modifications  Protein Folding: Many proteins require chaperones to fold correctly. Protease Cleavage: Inactive pre-proteins are activated by cleavage (e.g., neuropeptides). Sugars added to proteins in the ER and Golgi apparatus, crucial for receptor and membrane proteins. Phosphorylation: Addition of phosphate groups by kinases (e.g., RNA polymerase II phosphorylation). Ubiquitination: Targets proteins for degradation by the 26S proteasome. Ubiquitin: 76-AA protein is attached to target proteins at lysine residues for their degradation. Extracellular Signals Altering Translation Kinase Signaling:Phosphorylation cascades alter transcription factors to up/downregulate gene expression Regulation of mRNA: ○ Cap Removal: removing proteins that interact with the mRNA cap. ○ miRNA: Regulate gene expression by binding to 3' UTR of mRNA, affecting stability or translation. ○ Pausing elongation ○ Poly A binding proteins removal Protein Localization—key mechanisms 1. Nuclear Localization Signal: Nuclear Localization Signal (NLS) Proteins destined for nucleus contain an NLS that is recognized by cytoplasmic receptor proteins. Transport via nuclear pores allow large molecules to enter/exit the nucleus. 2. Signal Sequences for Organelle Targeting Signal: Short N-terminal leader peptide (signal sequence or signal peptide). Proteins destined for membranes or organelles have signal sequence at N-terminal end. signal sequence directs the protein to the appropriate channel for transport. Signal sequences are cleaved by peptidase after transport initiation. 3. Secretion Pathway Signal: N-terminal hydrophobic sequence. Hydrophobic signal sequence binds to proteins in the ER membrane. growing protein is threaded into the ER lumen. Signal sequence is cleaved by signal peptidase. 4. Processing in the Endoplasmic Reticulum (ER): Proteins are folded inside the ER. Ribosomes associated with ER ensure proteins are synthesized directly into the ER lumen. 5. Golgi Apparatus: Proteins are packaged into lipid vesicles for their final destination. Proteins are transported from the ER to the Golgi apparatus. 6. Final Destinations Membrane Proteins: Incorporated into the cell membrane. Secreted Proteins: Released extracellularly through vesicle fusion with the membrane. Gene Cloning :extracting a gene of interest from an organism’s DNA and making copies (clones) of it Identify the gene: Locate the gene of interest. Enzymes for Gene Cloning: 1. DNA polymerase: Involved in DNA replication.  2. DNA pol I: For processing DNA fragments. 3. Ligase: Joins DNA fragments. 4. Restriction Enzymes (Molecular Scissors): Cut DNA at specific sequences Example: Cloning the Insulin Gene 1. Extract insulin gene from pancreatic cells. 2. Convert RNA to DNA. 3. Amplify DNA using PCR. 4. Cut DNA using restriction enzymes. 5. Insert DNA into a vector (like a plasmid) for cloning. 6. Introduce plasmid into bacteria for replication. Amplifying a Gene of Interest 1. Restriction Enzyme Cloning: Cut the gene with restriction enzymes, isolate it, and insert into a vector. Use ligase to join DNA fragments. Use a plasmid with an origin of replication to allow bacterial replication of the inserted gene. 2. Polymerase Chain Reaction(PCR):In vitro (outside cell) amplification w/ specific primers to replicate gene. Requires knowledge of the sequence surrounding the gene of interest. Restriction Enzymes & DNA Cutting Recognition of Palindromic Sequences: Sticky Ends: staggered cuts (EcoRI). Blunt Ends: even on both sides. EcoRI Example: ○ Cuts DNA to produce sticky ends (single-stranded overhangs). ○ ligate sticky ends of different DNA fragments to form recombinant DNA. Directionality: Using 2 diff restriction enzymes like EcoRI and XhoI can control direction of ligation. Plasmids and Vectors—Types of Plasmids first Plasmids—Large plasmids used in gene cloning. Selectable Markers: ex. ampicillin resistance, to identify successful transformation in bacteria. Polylinker: A series of restriction enzyme sites used for easier insertion of DNA. Colony Screening: ○ Blue colony: No DNA insert (cleavage of X-gal). ○ White colony: DNA insert present (no cleavage of X-gal). Source of Thermostable Polymerases: 1. Discovery: In 1966, Thomas Brock found thermophiles in Yellowstone’s hot springs. 2. Thermophiles: Microorganisms with optimal growth at 60°C–108°C 3. Key Polymerases: critical for high-temperature PCR due to their thermostability ○ Taq Polymerase (Thermus aquaticus): Commonly used but error-prone. ○ Pfu Polymerase (Pyrococcus furiosus): Higher fidelity, processes larger DNA. Polymerase Chain Reaction (PCR) Inventor: Kary Mullis (1993 Nobel Prize). 1. Amplify target DNA region and add primers, dNTPs, and taq polymerase 2. Steps (repeated for amplification): Denature (95°C): Heat to separate DNA strands. Anneal (60°C): Cool to allow primers to bind to complementary sequences. Extend (72°C): Taq polymerase synthesizes new DNA strands.(optimal taq temp) 3. Cycle Repetition: Denature → Anneal → Extend repeated ~25 times. Amplification increases exponentially (~10⁶-fold after 25 cycles). Producing PCR Products with Sticky Ends 1. Initial PCR Steps: Heat to 95°C: Denature DNA to separate strands. Cool to Anneal: Allow primers to bind to target regions. Synthesize DNA: Taq polymerase extends the DNA strands. 2. Further Rounds of PCR: Continue cycles (~25–30 rounds) to exponentially amplify target DNA. 3. Digest PCR product with EcoRI: creating sticky ends for cloning. Ensure PCR primers include EcoRI recognition sites. ○ Verify target sequence doesn’t have EcoRI site bc digestion could cleave the fragment of interest Making RNA from Gene of Interest—in vitro transcription with T7 polymerase 1. Design Primers: forward primer containing T7 promoter sequence (18 bp: TAATACGACTCACTATAG) at 5' end followed by a sequence complementary to the target gene. reverse primer complementary to the opposite end of the target gene. 2. Set Up PCR:Denature, anneal, synthesize(multiple cycles to amplify the DNA) The forward primer will incorporate the T7 promoter into the DNA sequence at one end 3. Transcribe RNA: RNA polymerase will recognize T7 promoter and transcribe template into RNA Expressing Eukaryotic Genes in Bacteria Eukaryotic genes need to be converted into cDNA(complementary DNA) bc bacteria can’t process introns. Making cDNA: 1. Isolate mRNA: Extract mRNA from eukaryotic cells. 2. Reverse Transcription: Use reverse transcriptase (from viruses) to convert mRNA to cDNA. ○ Oligo-dT primers hybridize to the poly-A tail of mRNA. ○ Reverse transcriptase synthesizes the cDNA strand. 3. Synthesize Double-Stranded cDNA: Remove mRNA & synthesize 2nd DNA strand using DNA polymerase I Producing cDNA Molecules with Sticky Ends 1. Add Sticky Ends to cDNA: Ligate oligonucleotide linkers containing EcoRI to the cDNA. 2. Insert into Vector: Use EcoRI to insert the cDNA into a plasmid vector. 3. Transcription: IPTG to inactivate Lac repressor so T7 pol can transcribe cDNA and express gene in bacteria.  Purifying Protein: Histidine Tag (His-tag): Add sequence for 6 histidines to protein to purify w/ Ni2+-coated beads 1. Bind His-tagged proteins to Ni2+ beads. 2. Wash to remove unbound proteins. 3. Elute His-tagged protein using imidazole. Sequencing Genomes Fosmids and BACs (Bacterial Artificial Chromosomes): clone larger DNA fragments. human genome sequenced using large inserts to map the sequences across chromosomes. Modes of Delivering Recombinant DNA into Bacterial Cells: 1. Plasmids and BACs: Transformation (introduce DNA into bacteria). 2. Fosmids: Transduction (using phages to deliver DNA). 3. Bacteriophage Vectors: Infection followed by lysis to produce phage plaques. Confirming the Presence of the Insert 1. Grow Bacterial Colonies: Isolate colonies and test for plasmid presence. 2. Digest Plasmid with EcoRI: Confirm the presence of the gene insert by running on an agarose gel. 3. Hybridization (Southern Blot): Use a labeled probe to detect the DNA insert in colonies. Blotting Techniques: ○ Northern Blot: Detect RNA. ○ Southern Blot: Detect DNA. ○ Western Blot: Detect proteins. Restriction Enzyme-Independent Cloning Gibson Assembly: fast, cost-effective method for cloning multiple DNA fragments. ○ Can assemble multiple fragments in one reaction, no need for restriction enzymes. ○ Enzymes Used: 1. T5 Exonuclease: Chews back DNA ends to create overlaps. 2. DNA Polymerase: Fills in gaps. 3. DNA Ligase: Joins DNA fragments together. ○ Procedure: Mix fragments (including vector) and use enzymes to assemble into desired construct. Seamless Cloning: join DNA fragments in predetermined order w/out sequence restrictions or scars Synthetic biology (e.g., moving whole operons for metabolic engineering). Whole genome reconstructions. ○ Ex. 1: 2015: >20 genes from plants, mammals, bacteria, yeast. ○ Ex.2: 2010: Mycoplasma laboratorium genome. Sequencing DNA: uses base-pair complementarity and DNA polymerase 5' to 3' activity. main methods Dideoxy Sequencing (Sanger Sequencing), developed by Fred Sanger. Applied to bacterial virus phiX174 (smallest genome). Uses dideoxynucleotide triphosphates ( w/ missing 3' OH group) to terminate chain elongation.  Sanger Sequencing Steps ○ DNA template (PCR product, plasmid insert, genomic/cDNA). ○ Primer (3' OH for DNA synthesis). ○ Normal dNTPs (A, T, C, G). ○ A small amount of dideoxynucleotide (ddNTP: A, T, C, or G). 2. Reaction: ○ DNA synthesis with ddNTP incorporated at random, terminating chain elongation. ○ Separated by size on a gel (smaller fragments run further). 3. Reading: DNA sequence read from the bottom (smallest fragment) to the top (longest fragment). ○ Fluorescent ddNTPs: Use of fluorescently labeled ddNTPs (green, red, blue, yellow for A, T, C, G). ○ Sequence read from electrophoresis, detected by a laser and recorded by computer. Genome Sequencing Process 1. Fragmenting Genome: Cut the genome into random fragments and sequence each fragment. 2. Overlap & Contigs: Overlap sequence reads to assemble contigs (continuous sequences). ○ Method developed by Craig Venter (founder of Celera Genomics, sequenced human genome). Whole Genome Shotgun Sequencing (WGS): Sequence first, map later. Paired-End Reads: ○ Sequence both ends of a fragment to help assemble larger genomes. ○ End reads from multiple clones overlap to create a full genome. Next Generation Sequencing (NGS) Example: Illumina dye sequencing (many systems available). Key Features: ○ Sequencing without cloning into microbial hosts. ○ Parallel sequencing of millions of DNA fragments. ○ Short reads (