Unformatted text preview: Review LS3 weeks 1-3 (Lecture 10)
Big Picture Hereditary Material is DNA 3 experiments: Griffith Avery, Mcleod & McCarty Hershey-Chase Structure of DNA -Watson & Crick (and many others) Central Dogma of Molecular Biology DNA ----> RNA ----> protein DNA in the cell exists as Chromosomes - which contain Genes One Gene = one Polypeptide -- GENETIC CODE Big Picture What has been learned from the ‘model systems’? Genetics Correlate phenotype (visible/measurable character) with ‘gene’ and position of gene on chromosome. Protein Purification Purified protein yields polypeptide sequence from which gene sequence is inferred. Genomics Predict all genes in genome. Predict all proteins – inferred from DNA sequence. Repetitive DNA between genes. Big Picture What has been learned from the ‘model systems’? Proteomics Measure all proteins present in cell. Measure differences in proteins between different states of the cell. Major Topics The model systems Difference among Eukaryotes, Archea, & Eubacteria Be able to recognize the names of each model and type of organism Be able to recognize increasing complexity Be able to recognize the relative benefits of each system Major Topics Chemistry background Know: Polar structure, e.g. water
Hydrogen bond Hydrogen bonds Hydrophobic interactions Van der Waals forces 5Å Major Topics Discovery of DNA as Genetic Material Griffith experiment Transforming Principle killed Streptococcus polysaccharide capsule Avery, Macleod & McCarty experiment Transforming Principle is DNA Hershey-Chase Experiment 32P- and 35S-labelled bacteriophage Major Topics Structure of DNA What lead to Watson & Crick’s Discovery? Purines and Pyrimidines Deoxyribose Phosphate Major Topics Structure of DNA Nucleoside vs. nucleotide Nucleotide chains have 5’-3’ polarity – phosphodiester bonds Major Topics Structure of DNA Chargaff’s Rules double-stranded anti-parallel helix, Held together by: Inter-strand base pairing …… Intra-strand hydrophobic stacking Major Topics Structure of DNA B-form DNA Right-handed helix 10-bp per turn Face inwards Faces outwards A-form DNA Right-handed helix Major groove deep & narrow (also found in dsRNA) Major Topics Structure of DNA Major groove contains ‘information’ A = H-bond acceptor D = H-bond donor H = non-polar hydrogen M = hydrophobic methyl Potential for hydrogen bonding and hydrophobic interactions Major Topics Structure of DNA Denature/melting of strands breaking Hydrogen bonds Heat Chemicals Re-annealing of strands cooling salt mismatches Major Topics Topology of DNA Relaxed Supercoiled (negative vs. positive) Twist (# helical turns) + Writhe (# superhelical turns) = Linking number Major Topics Topology of DNA Topoisomerase I breaks one strand changes linking number by 1 Covalent linkage through tyrosine Topisomerase II breaks two strands changes linking number by 2 Major Topics Topology of DNA in Eukaryotic cell Chromosomes (long linear) are tightly wound 700 nm 200 nm loops 30 nm fiber 10 nm beads Major Topics Topology of DNA in cell Beads are called Nucleosomes Core Histones histone H3 and histone H4 tetramer 2x histone H2A and histone H2B Linker Histone histone H1 DNA wrapped around ~2 times Major Topics RNA structure Ribose has 2’ hydroxyl And uracil instead of thymine G:U wobble base-pairing allowed Major Topics RNA structure Much is single stranded dsRNA allows mispairing A-form helix – Right handed Bulges on one side of stem Loops -hairpin -internal Pseudoknot Major Topics Messenger RNA (mRNA) structure • Contains continuous open-reading frame (ORF) encoding a speciﬁc polypeptide UAA UGA UAG Stop = Poly(A) tail 5’-cap Coding region = AUG Start Major Topics Genetic code Determined in Nirenberg experiments In vitro translation with synthetic RNA templates Three possible reading frames mRNA is read by transfer RNA (tRNA) Know how to use codon table Major Topics Transfer RNA (tRNA) Reads mRNA through anticodon 3’ 5’ 5’ 3’ Major Topics Protein (polypeptide) structure Peptide bond between two amino acids Carboxyl group to Amino group Planar [inflexible] Amino Acid Amino Acids Know the properties (acidic, basic, polar, special) by name Major Topics Protein secondary structure α-helix • H-bond between Amino and Carboxyl groups of every 4th AA • 3.6 residues (AAs) per turn = ~7 AAs per two turns NB 5.4Å (not 4.5Å) • H-bonds are parallel to axis of the helix • R groups project out from helix Major Topics Protein secondary structure β-sheet -Planar peptide bonds with bend at Cα -H-bonding between Amino and Carboxyl groups from one beta strand to another -R groups alternate above and below sheet Major Topics Protein tertiary structure 3-D organization of secondary structures Stabilized by: disulphide bonds hybrophobic interactions Ionic interactions Major Topics Protein Quaternary structure 3-D organization of two, or more, molecules Homo-dimer -trimer -tetramer Hetero- Major Topics Protein Domain structure Classified by shape or function Globular fibrous/stalk DNA binding Small domain also called Motif (structural) zinc finger Sequence motif consensus amino acid sequence derived from multiple sequence alignment zinc finger C-X2-C-Xn-H-X3-H Major Topics Protein Interaction Domains Coiled coil – hydrophobic interaction between two α helices Keratin Hydrophobic @ 4 AA intervals (i.e. one turn) Leucine zipper Leucine @ 7 AA intervals in helix (i.e. two turns) Major Topics Post-translational modifications of proteins Proteolytic cleavage proteases self cleavage Phosphorylation Methylation Glycosylation Acylation Protein conjugation (ubiquitin) Major Topics Regulation of protein (enzyme) activity (1) Transcription Translation Folding Spontaneous Assisted – chaperones –- heat shock proteins & chaperonins – GroEL + ATP Major Topics Regulation of protein (enzyme) activity (2) Effector binding (allostery) Positive Negative Stability Degradation ubiquitin Proteasome Major Topics Antibodies IgG structure Monoclonal & Polyclonal Light & Heavy chains Y structure Fab and Fc fragments Antibody ‘sandwich’ for detection assays Western blot Immunofluorescence Function Neutralize proteins & microbes Target specifc cells, e.g. tumor cells Big Picture Protein purification Why would one want to purify a protein? To know how different phenotypes are caused To know the basis for biochemical pathways To understand the basis for disease To understand the function of all the proteins in a cell or different cells Major Topics Protein purification (preparative techniques) Based on properties of the protein that distinguishes it from other proteins Size Charge -size exclusion chromatography -ion exchange chromatography - overall positive or negative charge? Cation exchange = negative charged matrix -what does it like to bind to? Affinity Assay = means to measure presence of the protein Major Topics Protein Analytical Techniques SDS-Polyacrylamide Gel Electrophoresis Denaturing – SDS gives overall negative charge Reducing – mercaptoethanol 1D 2D Western blotting Major Topics Protein Sequence determination (1) Edman degradation - sequential Amino terminus 1. couple PITC 2. cleave with acid 3. detect PITC-AA by HPLC start again at 1 Overlapping proteolytic peptides to obtain complete sequence Trypsin leaves Arginine or Lysine as Carboxyterminal AA Major Topics Protein Sequence determination (2) Mass Spectrometry Peptide Mass Fingerprints compared to in silico database -requires pure protein -requires complete genome sequence Tandem Mass Spectrometry 1. protein sequence de novo 2. complex mixture of proteins with complete genome sequence Peptides are isolated and fragmented for mass/charge ratio determination ...
View Full Document
- Spring '09
- DNA, Major Topics Protein, Central Dogma of Molecular Biology DNA