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What is the flow of information through the cell? Double helix - antiparallel polymers Major groove Minor groove 5’ 3’ A Purine G T Pyrimidine C 06_12_asymmetrical.jpg Transcription: • dsDNA template • Nucleotides (ACGU) make ssRNA • Need to separate strands. • Nucleotides added to free 3’ OH (5’3’) Classes of RNA… mRNA rRNA …also snRNA and microRNA tRNA Prokaryotes untranslated regions 5’ UTR promoter (not transcribed) 3’ UTR coding sequence DNA mRNA RNA Polymerase Ribosome polypeptide Eukaryotes untranslated regions 5’ URT promoter (not transcribed) 3’ UTR coding sequence DNA pre mRNA mRNA RNA Polymerase Ribosome polypeptide Bacterial Promoter Elements • • • • Transcription start = +1 Consensus sequence = –35; TTGACA, recognized by Pribnow box = -10, TATAAT; determines +1 Terminator sequence: where polymerase stops Initiation of transcription in prokaryotes Initiation of eukaryotic transcription by RNA Pol II (mRNA) TF = transcription factor (compare with prokaryotic sigma factor) Eukaryotic mRNA: • 5’ cap, • 5’ UTR • coding region • 3’ UTR • 3’ poly-A tail mRNA processing in Eukaryotes 5’ cap added Remove 3’ end Poly-A tail added Introns removed Exons joined Gene cloning - making lots of copies... 1. 2. 3. 4. Make a “library” of small pieces of DNA (2 types) Find the one piece you want Insert it into a “vector” Grow it in a new organism (bacteria, euk. cells) replication transcription DNA translation RNA Isolate DNA, fragment with RE Genomic library Protein Isolate mRNA, convert to cDNA with reverse transcriptase cDNA library Overview of gene expression in eukaryotes 07_37_Protein.produc.jpg 07_28_ribosome.jpg Two adapters link an amino acid to a codon 07_26_2_adaptors.jpg Intiation of translation in Eukaryotes 07_32_initiation.jpg Intiation of translation in Prokaryotes 07_33_mRNA.encode.jpg Elongation of proteins 07_30_3_step_cycle.jpg 4 5 07_34_stop codon.jpg Termination of translation Mutations… • Frameshift: Adding or removing 1 or 2 nucleotides results in changes the reading frame from that point on. • Nonsense: Changing an amino acid codon to a stop codon results in truncated proteins • Missense: Changing an amino acid codon to one encoding a different amino acid - effect depends on type of amino acid and where in the protein. 04_03_20 amino acids.jpg Side chains interact via all of the noncovalent bonds Primary structure (1°) of a protein: Arabidopsis -glucosidase (single letter codes) MSSLHWFPNIFIVVVVFFSLRSSQVVLEEEESTVVGYGYVVRSVGVDSNRQVLTAKLDLIKPSSV YAPDIKSLNLHVSLETSERLRIRITDSSQQRWEIPETVIPRAGNHSPRRFSTEEDGGNSPENNFL ADPSSDLVFTLHNTTPFGFSVSRRSSGDILFDTSPDSSDSNTYFIFKDQFLQLSSALPENRSNLY GIGEHTKRSFRLIPGETMTLWNADTGSENPDVNLYGSHPFYMDVRGSKGNEEAGTTHGVLLLNSN GMDVKYEGHRITYNVIGGVIDLYVFAGPSPEMVMNQYTELIGRPAPMPYWSFGFHQCRYGYKNVS DLEYVVDGYAKAGIPLEVMWTDIDYMDGYKDFTLDPVNFPEDKMQSFVDTLHKNGQKYVLILDPG IGVDSSYGTYNRGMEADVFIKRNGEPYLGEVWPGKVYFPDFLNPAAATFWSNEIKMFQEILPLDG LWIDMNELSNFITSPLSSGSSLDDPPYKINNSGDKRPINNKTVPATSIHFGNISEYDAHNLYGLL EAKATHQAVVDITGKRPFILSRSTFVSSGKYTAHWTGDNAAKWEDLAYSIPGILNFGLFGIPMVG ADICGFSHDTTEELCRRWIQLGAFYPFARDHSSLGTARQELYLWDSVASSARKVLGLRMRLLPHL YTLMYEAHVSGNPIARPLFFSFPQDTKTYEIDSQFLIGKSIMVSPALKQGAVAVDAYFPAGNWFD LFNYSFAVGGDSGKHVRLDTPADHVNVHVREGSIVAMQGEALTTRDARKTPYQLLVVASRLENIS GELFLDDGENLRMGAGGGNRDWTLVKFRCYVTGKSVVLRSEVVNPEYASKMKWSIGKVTFVGFEN VENVKTYEVRTSERLRSPRISLIKTVSDNDDPRFLSVEVSKLSLLVGKKFEMRLRLT Secondary structure (2°) -helix H-bonds between C=O and N-H of backbone. (No R-groups involved) Secondary structure -sheet H-bonds between C=O and N-H of backbone. (No R-groups involved) Tertiary structure - the entire polypeptide -helix -sheet loops and turns disulfide bridge ribonuclease Quaternary structure multiple subunits e.g. hemoglobin cytochrome b562 lactic (lactate) dehydrogenase immunoglobulin light chain 04_20_protein domains.jpg Domains - discrete modules within tertiary structure that fold independently and have a specific function. 4 domains of phospholipase C Motif - a recurring substructure / barrel e.g. -amylase Motif - a recurring substructure coiled coil e.g. myosin How do proteins get to their folded state? unfolded native conformation Proteins with different functions may have similar shape members of a family with a common ancestor. 04_21_Serine proteases.jpg 04_22_protein subunit.jpg