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11 Introduction to Cells & Microscopy Introduction to Cells & Microscopy 5.1 What Features Make Cells the Fundamental Units of Life? 5.1 What Features Make Cells the Fundamental Units of Life? Microscopes: Magnification: Increases apparent size Resolution: Clarity of magnified object – minimum distance two objects can be apart and still be seen as two objects. Figure 5.3 Looking at Cells (Part 1) Two basic types of microscopes: Light microscopes—use glass lenses and light. Resolution = 0.2 µm Electron microscopes—electromagnets focus an electron beam. Resolution = 0.2 nm Figure 5.3 Looking at Cells (Part 2) Figure 5.3 Looking at Cells (Part 3) 1 5.1 What Features Make Cells the Fundamental Units of Life? 5.1 What Features Make Cells the Fundamental Units of Life? The plasma membrane: PARTS OF CELLS: • Is a selectively permeable barrier ALL CELLS • Allows cells to maintain a constant internal environment The plasma membrane is the outer surface of every cell. • Is important in communication and receiving signals The cytoplasm the inner contents of every cell. The cytoplasm: • Often has proteins for binding and adhering to adjacent cells • Is a concentrated aqueous environment (cytosol) • It is very crowded with high concentrations of molecules and proteins, some as a colloidal suspension Figure 5.4 A Prokaryotic Cell 5.1 What Features Make Cells the Fundamental Units of Life? Two types of cells: Prokaryotic and eukaryotic Bacteria and Archaea are prokaryotes. The first cells were probably prokaryotic. Eukarya are eukaryotes—the DNA is in a membrane-enclosed compartment called the nucleus. They also contain organelles such as mitochondria and chloroplasts. Cells are larger (>10,000 in volume) The Gram Stain and the Bacterial Cell Wall Figure 5.7 Eukaryotic Cells (Part 1) Outside of cell Cell wall (peptidoglycan) Gram Positive Plasma membrane 5 µm Inside of cell Outer membrane of cell envelope Outside of cell Periplasmic space Gram Negative Cell envelope Peptidoglycan layer Periplasmic space 5 µm Plasma membrane Inside of cell 2 12 Central Dogma of Molecular Biology Introduction to Cells & Microscopy From DNA to Protein: Gene Expression • Central Dogma: from Genes to Proteins • Replication of the genes • Transcribing the information • Translating the nucleotide sequence into protein sequence – The Genetic Code – Protein Biosynthesis • Control of gene expression – prokaryotic – eukaryotic Central Dogma The central dogma of molecular biology Replication 13 DNA and Replication Information Flow DNA and Its Role in Heredity • Evidence for the Gene and that it is made of DNA – Avery experiments – Hershey-Chase experiments • Structure of DNA – Franklin-Wilkins experiments – Watson-Crick structure – Messelson-Stahl experiments • DNA Replication • DNA Repair 13.1 What Is the Evidence that the Gene Is DNA? Frederick Griffith, determined that a transforming principle from dead cells produced a heritable change in the live cells….. What was this transforming principle? Oswald Avery (1944) treated samples to destroy different molecules; if DNA was destroyed, the transforming activity was lost. There was no loss of activity with destruction of proteins, carbohydrates, or lipids. 3 Figure 13.2 Genetic Transformation by DNA (Part 1) Figure 13.2 Genetic Transformation by DNA (Part 2) RNAse treated Figure 13.3 Bacteriophage T2: Reproduction Cycle Protease treated DNAse treated Figure 13.4 The Hershey–Chase Experiment (Part 1) Hershey-Chase experiment: • Used bacteriophage T2 virus to determine whether DNA, or protein, is the genetic material • Bacteriophage proteins were labeled with 35S; the DNA was labeled with 32P T2 phage (virus) life cycle Figure 13.4 The Hershey–Chase Experiment (Part 2) DNA and Its Role in Heredity • Structure of DNA – Franklin-Wilkins experiments – Watson-Crick structure – Messelson-Stahl experiments 4 Figure 13.6 X-Ray Crystallography Helped Reveal the Structure of DNA 13.2 What Is the Structure of DNA? • The crucial piece of evidence for DNA structure came from X-ray “crystallography.” Wilkins learned how to purify DNA and make regular fiber patterns. Rosalind Franklin performed the X-ray diffraction and deduced there was a helix. • Francis Crick saw the data at a seminar Wilkins gave and also deduced there was a helix and the size parameters. • James Watson discovered how the bases went together (complementarity) using Chargaff rules (A=T, G=C). Rosalind Franklin • Watson & Crick published their structure in 1953. Beautiful example of how structure predicted function. Figure 13.7 DNA Is a Double Helix (Part 1) Chargaff’s Rule Photo 13.2 Computer-simulated space-filling model of DNA. Video: Computer-simulated space-filling model of DNA. 5 Figure 13.7 DNA Is a Double Helix (Part 2) 13.2 What Is the Structure of DNA? Other aspects of DNA structure that became apparent: • Genetic material is susceptible to mutation—a change in information— possibly a simple alteration to a sequence. (34 Å)" sugar–phosphate backbone (phosphodiester bonds) Right-handed, antiparallel, doublestranded helix. With the “base complementarity,” it explains genetic material: • Storage of genetic information • Replication • Information retrival Experimental Proof of Structure: • Access to DNA by proteins is possible as a sequence-dependent binding interactions through major groove. Figure 13.11 The Meselson–Stahl Experiment (Part 1) Messelson and Stahl Experiment: Given the double-helix, how is DNA replicated? Three possible replication patterns: § Semiconservative: Parent serves as a template and new molecules have one old and one new strand § Conservative: Original helix only serves as a template § Dispersive: Parent fragments serve as templates, assembling old and new parts into molecules They used density labeling to distinguish parent DNA strands from new DNA strands. DNA was labeled with a heavy isotope, 15N, making it more dense. Figure 13.11 The Meselson–Stahl Experiment (Part 2) 13.3 How Is DNA Replicated? • DNA Replication Arthur Kornberg showed that DNA contains information for its own replication. He combined in a test tube: DNA, the four deoxyribonucleoside triphosphates (dNTPs–monomers), DNA polymerase, salts (Mg+2), and buffer. The DNA served as a template for synthesis of new DNA. 6 Figure 13.12 Each New DNA Strand Grows from Its 5´ End to Its 3´ End (Part 1) 13.3 How Is DNA Replicated? A large protein complex—the replication complex —does the work. Proteins in the Complex: s Helicase - unwinds double helix s Single-stranded binding protein - maintains ssDNA s Primase- makes the primer (small RNA) s DNA Polymerase III - does the synthesis s DNA Polymerase I - removes primer, fills in gaps s DNA ligase - makes final phosphodiester bond Start point: All chromosomes have a region called origin of replication (ori). Proteins in the replication complex bind to a DNA sequence in ori. Figure 13.14 DNA Polymerase Binds to the Template Strand (Part 2) The Replicaton Fork Replication Fork Leading strand 3 ’ DNA polymerases need a primer. RNA polymerase (primases) do not. 5 ’ Primer Primase 3’d5’ DNA helicase uses energy from ATP hydrolysis to unwind the DNA. Single-strand binding proteins keep the strands from getting back together. Lagging strand Figure 13.20 Telomeres and Telomerase (A) Problem #2: Synthesis of both strands What about the last Okazaki fragment at the end of the chromosome (the telomere)? Leading strand No synthesis of other strand!" Lagging strand No DNA synthesis occurs because there is no 3’ end to extend —a single-stranded bit of DNA is left at each end. If left, these single-stranded regions would be removed after replication; after many replications, continued chromosome shortening would lead to cell death. Telomere" Leading Parental Stand" Lagging Parental Stand" 3’-end" Looped around so that polymerase III can synthesize 5’ g 3’ from both strands…and in the same physical direction" Lagging strand is released when it gets too big (~2000 bp)" Okazaki fragments" New primer needed. Pieces of lagging strand called Okazaki fragments" Replication Fork Direction" 7 Figure 13.20 Telomeres and Telomerase (A) What about the last Okazaki fragment at the end of the chromosome (the telomere)? No DNA synthesis occurs because there is no 3’ end to extend —a single-stranded bit of DNA is left at each end. If left, these single-stranded regions would be removed after replication; after many replications, continued chromosome shortening would lead to cell death. Solution: 1) Telomeres– repetative DNA 2) Telomerase– fill in using self-contained primer to repeated DNA 13.3 How Is DNA Replicated? Eukaryote chromosomes have repetitive sequences at the ends called telomeres. These repeats are protective and prolong cell division, especially in rapidlydividing cells, like bone marrow. Telomerase contains an RNA sequence —acts as template for telomeric DNA sequences. Figure 13.20 Telomeres and Telomerase (B) DNA and Its Role in Heredity • DNA Repair 3’d5’ Telomeric DNA filled in ends Figure 13.21 DNA Repair Mechanisms (A) 13.4 How Are Errors in DNA Repaired? DNA polymerases make mistakes in synthesis (1/10,000), plus DNA can be damaged in living cells. Cells have three repair mechanisms: • Proofreading (1/10,000) • Mismatch repair (1/100) • Excision repair correct base wrong base Phosphodiester bond made Phosphodiester bond hydrolyzed Detected by 3’ à 5’ exonuclease activity (part of DNA polymerase) Phosphodiester bond made 8 Figure 13.21 DNA Repair Mechanisms (B) Figure 13.21 DNA Repair Mechanisms (C) The newly replicated DNA is scanned for mistakes by other proteins. A mismatch repair mechanism detects mismatched bases—the new strand has not yet been modified (e.g., methylated in prokaryotes) so it can be recognized. If mismatch repair fails, the DNA is mutated. 14 DNA can be damaged by radiation, chemicals in the environment, and random spontaneous chemical reactions. Excision repair: Enzymes constantly scan DNA for damaged bases—they are excised and DNA polymerase I adds the correct ones. 14.2 How Does Information Flow from Genes to Proteins? Gene Expression: Transcription and Translation Central Dogma RNA is key to this process: • Messenger RNA (mRNA)—carries copy of a DNA sequence to site of protein synthesis at the ribosome • Transfer RNA (tRNA)—carries amino acids for polypeptide assembly • Ribosomal RNA (rRNA)—catalyzes peptide bond formation and provides structure for the ribosome Central Dogma The central dogma of molecular biology Replication Messenger RNA (mRNA) Transfer RNA (tRNA) Ribosomal RNA (rRNA) • How does genetic information get from the nucleus to the cytoplasm? Francis Crick proposed TWO hypotheses, based on their structure of DNA: • Messenger hypothesis—a complementary copy of one DNA strand of the gene is made. The “transcript” travels from nucleus to cytoplasm carrying information as codons (packages of information encoding the protein). • Adapter hypothesis—an adapter molecule exists in the cell that can bind amino acids, and recognize a nucleotide sequence, or these “codons.” These adapter molecules must contain anticodons complementary to these codons, their recognition based on the complementary base pairing found in the DNA. During protein biosynthesis, these adaptor molecules carry amino acids in proper sequence to interpret or decypher the sequence of the polypeptide chain—translation. • What is the relationship between a DNA sequence and an amino acid sequence? 9 Central Dogma: in Eukaryotes Prokaryotes and eukaryotes differ in execution of the Central Dogma Transcription Transcription components: • A DNA template for base pairings—one of the two strands of DNA • Nucleoside triphosphates (ATP,GTP,CTP,UTP) as substrates • An RNA polymerase enzyme Transcription: Initiation Transcription Transcription occurs in three phases: • Initiation • Elongation • Termination Transcription: Initiation Initiation requires a promoter—a special sequence of DNA. RNA polymerase binds to the promoter. Promoter tells RNA polymerase where to start and which strand of DNA to transcribe. Part of each promoter is the initiation site. 10 Transcription: Elongation Transcription: Elongation Elongation: RNA polymerase unwinds DNA about ten base pairs at a time; reads template in 3’ to 5’ direction, synthesizes RNA in the 5’ to 3’ direction. The RNA transcript is antiparallel to the DNA template strand, and adds nucleotides to its 3’ end. NTPs incorporate NMP and PPi is a product! RNA polymerases do not proofread and correct mistakes (so only initial error rate of 1/10,000). Transcription: Termination Transcription: Termination Termination: Is specified by a specific DNA base sequence. Mechanisms of termination are complex and varied. For some genes the transcript falls away from the DNA template and RNA polymerase—for others a helper protein pulls it away. Gene Structure Gene Structure In prokaryotes transcription and translation are done at the same place and time. In eukaryotes a nucleus separates transcription and translation. In prokaryotes, many genes are organized in polycistrons. Geneall A genes are Gene B Gene CAlso, they In eukaryotes, monocistronic. may promoter terminator have noncoding sequences, called introns, with the coding sequences called exons. Initiation of transcription" 5’-flanking End of transcription" 3’-flanking Cistrons all have one promoter toGene which RNA polymerase binds (with the promoter terminator help of other molecules), andexon at theexon other end exon exonof the gene, a terminator intron intron polymerase intron sequence which signals where RNA should end transcription. 11 Post-transcriptional Modification Post-transcriptional Modification RNA splicing removes introns and splices exons together. Introns interrupt, but do not scramble, the DNA sequence that encodes a polypeptide. Sometimes, the separated exons code for different domains (functional regions) of the protein. Consensus sequences are short sequences between exons and introns. snRNPs binds here, and also near the 3′ end of the intron. In the nucleus, pre-mRNA is modified in TWO ways at each end: 1) The 5’ end is “capped.” G cap (modified guanosine triphosphate) is added. The cap protects mRNA from being digested by ribonucleases and facilitates binding to ribosome Stop Codon" during translation. Start Codon" 3’-Untranslated Region (3’-UTR)" 5’-Untranslated Region (5’-UTR)" Exon 1 intron Exon 2 Exon 1 Exon 2 intron Exon 3 Exon 3 Open Reading Frame (ORF)" Translation: The Genetic Code (Open Reading " Frame (ORF)" 20-21 Pol-A " site" bases" { Newly transcribed pre-mRNA is bound at ends by snRNPs—small nuclear ribonucleoprotein particles. Pol-A signal" 5’-UTR" 3’-UTR" 2) The 3’ end is poly-adenylated. A Poly-A tail is added 20-21 bases after the poly-A “signal” (AAUAAA) sequence, which is after last codon. This sequence signals a nuclease to cut the pre-mRNA; then another enzyme adds 100 to 300 adenines. May assist in export from nucleus; important for stability of mRNA. Translation: The Genetic Code The genetic code is redundant. The genetic code is universal. The genetic code: Specifies which amino acids will be used to build a protein Codon: A sequence of three bases—each codon specifies a particular amino acid. Start codon: AUG—initiation signal for translation. Stop codons: UAA, UAG, UGA—stop translation and polypeptide is released. Translation: tRNA Translation: tRNA tRNA, the adapter molecule, links information in mRNA codons with specific amino acids. For each amino acid, there is a specific type or “species” of tRNA. 12 Translation: tRNA tRNAs must deliver amino acids corresponding to each codon The conformation (three-dimensional shape) of tRNA results from base pairing (hydrogen bonding) within the molecule. 3‘-end is the amino-acid attachment site—binds covalently. At the other end (middle of the tRNA sequence) is the Anticodon—site of base pairing with mRNA. Unique for each species of tRNA. Translation: tRNA Example: 5’-CGG-3’ Coding strand (Crick; sense) DNA codon for arginine: 3’-GCC-5’ Template strand (Watson; antisense) Complementary mRNA: 5’-CGG-3’ Has the actual Codons Anticodon on the tRNA: 3’-GCC-5‘ This tRNA is charged with arginine. Antisense to the Codons For some tRNAs, there are multiple codons; e.g., that for alanine, GCA, GCG, GCC, and GCU. These are recognized by the same tRNA. This is possible due to Wobble: lack of specificity for the base at the 3‘-end of the codon (5‘–end of the anticodon in the tRNA). Wobble base Translation: tRNA Translation: Protein Biosynthesis Aminoacyl-tRNA synthetases—charge tRNA with the correct amino acids. Each enzyme is highly specific for one amino acid and its corresponding tRNA; the process of tRNA charging is called the second genetic code. The enzymes have three-part active sites: They bind a specific amino acid, a specific tRNA, and ATP. Translation: Protein Biosynthesis Ribosome: the workbench—holds mRNA and charged tRNAs in the correct positions to allow assembly of polypeptide chain. Ribosomes are not specific, they can make any type of protein. Translation: Protein Biosynthesis: Ribosome Structure Ribosomes have two subunits, large and small. When not active in translation, the subunits exist separately. • The small subunit (40S) has one ribosomal RNA (rRNA) (18S) and 33 proteins. • The large subunit (60S) has three molecules of rRNA (28S, 5.8S, 5S) and 49 different proteins. • Ribosomal subunits are held together by ionic and hydrophobic forces (not covalent bonds) (80S). 13 Translation: Protein Biosynthesis Translation: Protein Biosynthesis; Initiation Like transcription, translation also occurs in three steps: • Initiation • Elongation • Termination Translation: Protein Biosynthesis; Initiation Initiation: An initiation complex forms—a charged tRNA and small ribosomal subunit, both bound to mRNA: at the correct spot (AUG) • In prokaryotes, rRNA binds to mRNA recognition site “upstream” from start codon (Shine-Delgarno sequence). • In eukaryotes, the small subunit binds to the 5‘G-cap on the mRNA and moves until it reaches the start codon (Kozak rule). Translation: Protein Biosynthesis; Elongation Elongation: The second charged tRNA enters the A site. Large subunit catalyzes the two-part peptidyltransferase reaction: Ternary complex – It breaks bond between tRNA in P-site and its amino acid. – Peptide bond forms between that amino acid’s (or later that peptide) carboxyl and the amino acid on tRNA in the A-site (amino). IF2 GTP The rRNA has this activitiy…..catalytic RNA Translation: Protein Biosynthesis; Elongation Translation: Protein Biosynthesis; Elongation EF-Tu Translocation (GTP hydrolysis) GTP Decoding (GTP hydrolysis) Peptidyltransferase 14 Translation: Protein Biosynthesis; Elongation Translation: Protein Biosynthesis; Elongation – REVIEW When the first tRNA has released its methionine, as well as subsequent tRNAs, it moves to the E site and dissociates from the ribosome—can then become charged again. Elongation has these steps repeated (decoding, peptidyltransferase, translocation), assisted by proteins called elongation factors: – EF-Tu/EF-Ts for decoding – EF-G for translocation These steps require energy (2GTP). © One at decoding in the A-site. © One at translocation. Translation: Protein Biosynthesis; Termination Translation: Protein Biosynthesis; Termination Termination: Translation ends when a stop codon enters the A site. Stop codon binds a protein release factor (termination factor)—allows hydrolysis of bond between polypeptide chain and tRNA on the P site. Polypeptide chain separates from the ribosome—C terminus is the last amino acid added. Translation: Protein Biosynthesis; Termination Translation: Protein Biosynthesis; Termination 15 Translation: Protein Biosynthesis Post-translational Events Post-translational Events Post-translational aspects of protein synthesis: 1) FOLDING: Polypeptide emerges from the ribosome and folds into its 3-D shape. 2) LOCATION: It may contain a signal sequence indicating where in the cell it belongs; ER, Nucleus, lysosome, etc. 3) PROCESSING: post-translational changes in covalent bonds Post-translational Events Post-translational Events Protein modifications: • Proteolysis: Cutting of a long polypeptide chain into final products, by proteases • Glycosylation: Addition of sugars to form glycoproteins, by glycosyltransferases • Phosphorylation: Addition of phosphate groups catalyzed by protein kinases— charged phosphate groups change the conformation 16 Post-translational Events Control of Gene Expression: Prokaryotes Example: The lac operon – an inducible system (polycistronic) RNA polymerase Repressor gene promoter operator Operon genes Degradation of lactose is a catabolic pathway: genes needed are induced—turned on when substrate is available. Control of Gene Expression: Prokaryotes Control of Gene Expression: Prokaryotes Start of Transcription The lac operon is an inducible system. Other E. coli systems are repressible— repressed when small molecules called corepressors bind to their repressors. Binding of co-repressor (effector) to repressorgrepressor changes shapegbinds to operatorginhibits transcription If environment changes and effector is used upgconc. dropsgco-repressor dissociates from repressorgrepressor changes shapegdissociates from operatorg transcription starts (replenishes effector) Control of Gene Expression: Prokaryotes Control of Gene Expression: Eukaryotes? SUMMARY: Inducers don’t bind DNA when effector binds-ON Repressors bind DNA when effector binds-OFF 17 Control of Gene Expression: Eukaryotes Eukaryotic gene regulation can occur at multiple points in transcription and translation: • Inititation of transcription w Chromatin remodeling (1) w Activation of transcriptional initiation (2) • • • • • • mRNA processing (3) mRNA transport (4) mRNA stability (5) Initiation of translation (6) Post-translational controls (7) Protein stability (8) 18