Cell bio exam 2 study guide PDF

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Jennifer mierisch cell bio exam 2 study guide ...

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DNA and DNA replication - Structure is 2 antiparallel strands held together by H-bonds (phosphodiester) - Sugar phosphate backbone - Sequence written 5’ (free phosphate group) to 3’ (free -OH group) - Held together by phosphodiester bonds - Phosphate groups carry a negative charge - Form double helix with 10 base pairs/turn - Complementary base pairing: A-T (2 H-bonds) & C-G (3 H-bonds) - 5’-GTCAAGT-3’ - 3’-CAGTTCA-5’ - Strongest binding is at C and G - Chargaff’s rule: number of purines (A, G) = number of pyrimidines (C,T) - Double helix: bases extend perpendicular to the backbone, proteins primarily interact with the major groove (more availability than minor groove, more accessible) - Genetics terminology - Genome: all the DNA in an organism - Gene: segment of DNA that has directions for making RNA or a protein - Allele: one of a variety of a form of gene, 1 from each parent - Chromatin: DNA and its associated proteins Components of chromosomes - Replication origin: site where DNA replication begins - Centromere: holds 2 chromosomes together to make sure each cell gets 1 copy during cell division - Telomere: contains repeated nucleotide sequences required for proper replication and protects ends of chromosomes - Recognized by proteins that recruit telomerase to help with DNA replication - Replication: telomerase binds to template strand, adds additional telomere repeats to template strand, completion of lagging strand by DNA polymerase - Structure - Interphase chromosomes are loosely packed, while mitotic chromosomes are condensed - The condensed state of mitotic chromosomes ensures separation of chromosomes during mitosis - In decondensed states, things get tangled up easily and might not separate equally - DNA packaging - Wraps around histones, packs - Histones associate with DNA, forming chromatin - Histones promote chromosome packing via nucleosomes - Chromatin fiber of packed nucleosomes folds into loops - Nucleosomes consist of histones and DNA - “Beads on a string”

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- Connected by linker DNA - Held together by noncovalent interactions - 8 subunits: 2 H2A, 2 H2B, 2 H3, 2 H4 - DNA wraps/unwraps 4 times/sec to allow for replication Histone acetylation and packing - Lysine (+) on tails of histones are acetylated - Helps DNA pack by connecting with negatively charged sugar phosphate backbone - Less packed = histone acetylation (no ionic bonds), transcription - More packed = histone deacetylation, (ionic bonds) more condensed

Heterochromatin vs. euchromatin - Euchromatin: less condensed DNA, gene rich - Heterochromatin: more condensed DNA, enriched at centromeres and telomeres - Contains very few genes, usually turned off - Converting from euchromatin to heterochromatin - Development, modification of histone tails - Genes we don't use are turned off and packed up - Heterochromatin formation - H3K9 methylation → attracts heterochromatin-specific proteins, modifies nearby histones, spreads until it encounters a barrier DNA sequence - Inappropriate spreading: beta-globin gene + anemia - Important in X-inactivation DNA replication - Each strand acts as a template - Semi-conservative: one original strand and one new strand - Utilizes DNA polymerase and other enzymes - Opening the double helix - DNA helicase unwinds the double helix, using energy from ATP - Single-strand DNA-binding proteins stabilize single-stranded DNA - DNA topoisomerases allow rotation of the helix to relieve strain during replication, and prevent tangling of DNA - Primers - DNA replication requires a primer for initiation (eukaryotes) - DNA primase synthesizes RNA primers for initiation of DNA replication - RNA primers serve as red flags to ensure extra proofreading of fragments - DNA polymerase - Clamp loader helps load on the sliding clamp onto DNA - DNA polymerase associates with clamp, ring slides along DNA as polymerase moves - Sliding clamp keeps DNA polymerase on DNA until it runs into double strand and falls off - New clamp would assemble for the next Okazaki fragment

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Synthesis and polymerase - RNA primer provides 3’ OH site for DNA synthesis - Nucleotides add in the 5’ to 3’ direction - Reaction is driven by hydrolysis of the pyrophosphate - When the pyrophosphate is released, polymerase moves on 1 nucleotide to the next reaction Replication fork - Both strands act as a template for synthesis in the 5’ to 3’ direction - Leading strand is synthesized continuously, while lagging strand is synthesized discontinuously as Okazaki fragments - Lagging strand synthesis: stops when it runs into the 5’ end of another fragment, RNA primer is degraded, sequence filled in by DNA polymerase DNA ligase - Enzyme that joins 3’ end of one fragment to the 5’ end of another Okazaki fragment - Uses 1 molecule of ATP to catalyze this otherwise unfavorable sealing reaction Polymerase ensures proper DNA synthesis - Correct nucleotide pairing is more energetically favorable than incorrect ones - Conformational change ensures addition of right pairing - Has 3’ to 5’ proofreading activity: exonucleolytic

Why not 3’ to 5’ synthesis? - Energy comes from 5’ end for phosphodiester bond - Triphosphate group is the source of energy - 3’ to 5’ would not work because DNA strand could lose triphosphate group necessary to form phosphodiester bond - It would also be unable to correct errors - There would be no energy available for correction Efficiency of RNA synthesis - Some genes will have greater expression than others - Quick release of RNA from DNA allows multiple copies of RNA to be made in a short amount of time Structure of RNA - Ribose, mostly single-stranded - A, G, C, U - Folds into particular shapes using complementary base pairing Transcription - Small piece of DNA acts as template - Either strand can act as a template - Brief complementary base pairing between template strand and ribonucleotides - Synthesized 5’ to 3’ but does not require a primer

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Read 3’ to 5’ When genes are transcribed they begin using a promoter RNA polymerase uses cofactors to unwind DNA - RNA polymerase I: most rRNA - RNA polymerase II: all protein-coding genes, plus genes for other noncoding RNAs (spliceosome) - RNA polymerase III: tRNA genes, 5S rRNA gene, small RNAs Prokaryotic transcription - Does not need additional factors to initiate transcription - Has promoter regions - Sigma factor: recognizes the promoter - Peptide subunit of RNA polymerase (needs it to recognize transcription)

Types of RNA - mRNA: intermediate between DNA and protein, complementary to DNA, short-lived - rRNA: structural support, enzymatic activity, can live for weeks - tRNA: adaptor molecule, translator of gene into amino acid information, lives for weeks Eukaryotic initiation of transcription - TATA box, BRE elements, INR, DPE (downstream promoter elements) - Upstream = before tra start point, downstream = after tra start point - Step 1 - TATA box: important promoter element - TBP causes distortion of TATA DNA which marks the site of the beginning of transcription - Step 2 - TFIID: TBP binds TATA box, TAF subunits recognize other sites near start site - Step 3 - TFIIB: recognizes BRE, recruits and positions RNA polymerase at start - Step 4 - TFIIF: stabilizes TFIIB/D/polymerase interactions and attracts TFIIE and TFIIH - TFIIE attracts TFIIH - Step 5 - TFIIH unwinds DNA (like helicase) - Phosphorylates Ser5 of RNA polymerase CTD and releases polymerases from the promoter Exon: expressed sequence Intron: intervening sequence RNA polymerase II: factory - Phosphorylation of Ser5 alone recruits capping factors - Phosphorylation of Ser2 and Ser5 recruits splicing proteins → allow for splicing binding factors

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Phosphorylation of Ser2 alone recruits 3’ end processing proteins and factors required for addition of poly-A tail

RNA capping (mRNA) - Need to add 5’ cap to RNA transcript - Steps - Phosphatase removes more terminal phosphate group - Guanylyl transferases adds a guanine - Methyl transferase adds a methyl group to the base - Forms cap - Cap modification occurs on mRNA Gene structure and splicing - Prokaryotes do not do splicing (no introns/exons) - Splicing: joining exons together and removing introns - Specific sequences in the intron are recognized by splicing machinery - 5’ end of intron and 3’ end, central adenine RNA splicing - Spliceosome is assembled as transcription occurs - Before RNA leaves the nucleus - Adenine in intron attacks 5’ splice site (covalent bond) - Cut 5’ forms a loop - Free OH end reacts with 5’ end of next exon, releasing a lariat Splicing machinery - snRNAs: small nuclear RNAs mediate key steps in splicing - complex w/protein subunits forming snRNPs - snRNPs: small nuclear riboproteins, spliceosome is formed by assembly of multiple snRNPs Splicing - BBP/U2AF check branchpoint A sequence and U1 snRNP checks the 5’ splice site - U2 snRNP displaces BBP and U2AF to double check branchpoint A site - U4/U6/U5 enter to position mRNA for phosphoryl transfer reactions - U1 is replaced by U6, and U4 leaves - Lariat forms between first nucleotide of intron and branchpoint A via phosphoryl transfer reaction - 2 exons are joined together via 2nd phosphoryl transfer reaction Binding events - Binding by U1 snRNP and U6 ensures proper splice site selection - 2 phosphoryl transfer reactions form lariat and join exons - Binding by BBP and U2 snRNP ensures splicing accuracy

Recognition of processing signal - Dephosphorylation at Ser5 OR when Ser2 alone is phosphorylated recruits 3’ end processing proteins - Cleavage stimulating factor(CstF) and cleavage and polyadenylation specificity factor (CPSF) are bound to RNA polymerase tail - CstF and CPSF recognize and bind to a specific processing 3’ sequence - CPSF binds AAUAAA - CstF binds GU-rich element - Both are consensus sequences for polyadenylation - Cleavage and polyadenylation - CstF and CPSF assemble additional factors required for cleavage - RNA is cleaved - poly-A polymerase (PAP) adds about 200 nucleotides to the cleaved 3’ end - Poly-A bind proteins bind the poly-A tail - poly-A binding proteins determine the length of the tail and direct protein synthesis Eukaryotic mature mRNA - Has undergone capping, splicing, and polyadenylation - mRNA export from the nucleus - Smaller molecules can move through the pore freely - Larger molecules need to bind to nuclear transport receptor to help it move in/out (including mRNAs) Transcription of rRNAs - RNA polymerase I transcribes rRNAs - Does not have a C-terminal tail, therefore rRNAs are not capped or polyadenylated - Cell contains many copies of DNA that encode rRNAs, so a sufficient number of ribosomes can be produced - 3 RNAs (18S, 5S, 28S) are derived from a single precursor, 4th derived elsewhere - Assembled into ribosomes in nucleolus Translation - Genetic code - Translation of nucleotide sequence to amino acid sequence - mRNA nucleotide sequence read in 3’s - Codon: 3 consecutive nucleotides - 64 possible codons for 20 amino acids - Code is degenerate (more than 1 codon/amino acid) - Open reading frames (ORFs) - Continuous nucleotides that encode a protein w/no stop codons - 3 possible ORFs but only 1 encodes the protein, depends on where start codon (AUG) and stop codon are

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Adaptor for translation: tRNA - Small RNA molecules with frequent modification of nucleotides - Cloverleaf folds to form an L structure - Most important regions are anticodon (pairs with complementary mRNA) 3’ end where amino acid binds - Amino-acyl tRNA synthetase: catalyzes addition of the correct amino acid to the appropriate tRNA (high-energy bond, tRNA is charged) Anticodon base pairing and the wobble position - Wobble base can pair with multiple bases in the anticodon position, contributes to variability Ribosomes - help make proteins - Catalytic machine composed of rRNAs and proteins - Subunits come together on mRNA and form 4 binding sites - Small subunit provides for codon/anticodon pairing - Large subunit catalyzes peptide bond formation - Amino-acyl - tRNA, peptidyl - tRNA exit Initiation of translation (eukaryotes) - mRNA needs to have the 5’ cap and poly-A tail for translation - eIF4G/E associated with cap and tail (E before G, cap before tail) - Starts at AUG start codon Translation steps - 1. mRNA binds eIF4E at 5’ cap and eIF4G at poly-A tail - 2. mRNA binds to complex including the small ribosomal subunit, charged tRNA with methyl and eIF2 (with GTP) - 3. Initiator complex scans mRNA for start codon (AUG) - 4. When start codon is encountered, GTP on eIF2 is hydrolyzed to GDP and eIF2 is released, allowing the large ribosomal subunit to bind - 5. New charged tRNA enters at A site, growing polypeptide is held at P site, and uncharged tRNA is released from E site. ef-TU is released after GTP is hydrolyzed, ef-TU comes in and holds amino acid out of position, allowing time for proofreading - 6. Once an amino acid is added, the large ribosomal subunit shifts first - 7. EF-G (and GTP) will bind to the ribosome and assist in shifting the small ribosomal subunit - 8. Shifting continues until stop codon, when a release factor triggers dissociation of complex and release of polypeptide Elongation factors - ef-TU holds tRNA in a conformation, giving small ribosomal subunit time to proofread codon and anticodon - Released after hydrolysis of GTP - EF-G comes in bound to GTP, shifts ribosomal subunit along Termination of translation - Stop codons: UAA, UAG, UGA - Release factor binds A site

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- Water is added, releasing peptide chain - mRNA released and ribosomal subunits dissociate Polyribosomes - Lined up on mRNA - Increased protein production efficiency Protein folding: chaperones - Molecular chaperones bind partially folded polypeptides - Minimize energy used for folding - Prevent inappropriate association of unfolded proteins - Small: Hsp70 - Large: Hsp60 Protein degradation and proteasome - Polyubiquitin chain marks proteins for degradation - Sends proteins to proteasome (rich in proteases) - Proteases degrade protein - Proteasome cap recognizes protein to be degraded and helps thread it into the core (requires ATP) - Ubiquitination - Requires E1, E2, E3 - E1: ubiquitin activating enzyme - E2: ubiquitin conjugating enzyme - E2/E3: ubiquitin ligase - Ubiquitin ligases recognize degradation signals that are exposed as a result of protein misfolding - Ubiquitin is bound to the Lys of the target protein - Polyubiquitination targets the protein to the proteasome for degradation

Analyzing DNA - Polymerase chain reaction - Reagents, replicates DNA - Template DNA is added first, uses deoxynucleotides, DNA polymerase, primers - Uses changes in temperature - Separate 2 strands using heat (95 degrees Celsius) to break H-bonds - Use primers and cool reaction to bind to DNA, add DNA polymerase and deoxynucleotides - Also called amplification - See gene of interest with more cycles performed - Gel electrophoresis - Compares sizes of DNA fragments, agarose gel and anode - Small fragments migrate faster toward + electrode down - Larger move more slowly - Add chemical excited by UV light - DNA sequencing (Sanger)

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Sequences smaller strands to see what the nucleotides are DNA fragment and primer and polymerase, along with modified small deoxynucleotides (chain-terminating, labeled to know what last nucleotide is) that don’t have a hydroxyl group at 3’ carbon - DNA products loaded onto capillary gels -- every nucleotide and dideoxynucleotide is read to tell us the sequence Next generation DNA sequencing - Sequence an entire genome - Like Sanger, but no capillary gels - Uses specialized reagents and chain-terminating nucleotide - Different DNAs on slider plate - Removes terminator to add next nucleotide to keep reading - Removes fluorescent tag → to not mix signals

Membrane structure and function - Receive information - import/export of small molecules - Capacity for movement/expansion - Structure - Lipids and proteins interacting noncovalently - Lipid bilayer to protect hydrophobic tails - Dynamic structure -- can move around, fluid Types of lipids - Phospholipids: amphiphilic, 1 saturated and 1 unsaturated fatty acid (double bond) - Glycerol, 2 hydrocarbon tails (hydrophobic), hydrophilic head - Cholesterol: rigid structure, amphiphilic, reduces membrane permeability - Serine, hydrocarbon tail and hydrophilic head, polar head group - Rings interact with hydrocarbon chains - Role in fluidity is temperature dependent - High temperature keeps it rigid - Low temperature keeps it fluid - Glycolipids - Sphingosine - Sugars make up hydrophilic polar head group Lipid packing - Phospholipids typically form lipid bilayer -- keeps fatty acids safe - Hydrophilic heads face water, hydrophobic tails associate - Forms a lipid bilayer - Formation of a sealed compartment shields hydrophobic tails from water → energetically favorable Mobility in the plasma membrane

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Monolayers are called leaflets Lateral diffusion (within leaflet) is common Transverse movement (one leaflet to another) is rare

Properties of membranes - Unsaturated hydrocarbon chain with cis-bonds = more fluid - Reduce membrane thickness - Shorter hydrocarbon chains makes membrane more fluid due to fewer interactions Fluidity is temperature-dependent - High temperature = more fluid - Low temperature = less fluid Fluid mosaic model - Components of plasma membrane - Glycolipids, cholesterol, phospholipids - Proteins - Constantly moving Evidence for diffusion in the membrane - Fusion of cells reveals diffusion of proteins in the membrane - Mouse and human cells mixed, rapid movement in cell Fluorescence recovery after photobleaching (FRAP) - Used to measure the rate of diffusion of lipids and proteins in the membrane - Higher rate of diffusion = quicker rate of fluorescence return Restricting mobility in the membrane - Proteins aggregate - Tethered inside or outside the cell - Interact with proteins on other cells - Tight junctions: form between neighboring cells and stitch them together, keeps proteins restricted to different regions of the cell - Cytoskeleton domains: restrict proteins so they cannot move past the domain Phospholipid synthesis - Occurs in the ER - Scramblases randomly distribute phospholipids into the bilayer - Flippases (outer to inner) and floppases (inner to outer) sort to cytosolic and non-cytosolic leaflets Membrane proteins - Perform tasks in the membrane - 50% of membranes by mass, but not #’s due to large size compared to lipids

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Amounts and types vary in different cells - Integral membrane proteins: transmembrane proteins, located in one leaflet (lipid-linked or monolayer-associated) - Peripheral membrane proteins: bound to one leaflet, protein-attached

Transmembrane alpha helices - Can be single-pass or multipass - Single-pass: 1 alpha helix, hydrophobic regions interact with interior of the membrane, while hydrophilic regions are exposed on both sides, mostly nonpolar amino acids, NO PROLINE, ~20-30 amino acids - Multipass: more than 1 alpha helix, may have a mix of hydrophobic and hydrophilic or be entirely hydrophobic, depending on structure - Predicting membrane spanning alpha helices - Positive value means hydrophobic - Need 20-30 amino acids to span membrane as alpha helix Beta barrels form transmembrane channels - H-bonding between beta strands - Cannot be identified by hydropathy plot since only 10 amino acids required for beta strand to cross the membrane - Every other amino acid is hydrophobic, NO PROLINES interior - Rigid structure - Can form pores, with polar side chains facing inward Modification of membrane molecules - Oligosaccharides added to non-cytosolic side of lipids and proteins - Cells have a carbohydrate-rich layer on the outside known as the glycocalyx Permeability of lipid bilayer - Hydrophobic molecules: O2, CO2, N2, steroid hormones - Small uncharged polar molecules: H2O, urea, glycerol - Large uncharged polar molecules: glucose, sucrose - Ions: ...