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notes May 24 and 26.doc.doc

  1. 1. May 24, 2006 Fig 32-6: Genetic Map of bacteriophage x174 as determined by DNA sequence analysis. -have overlapping genes -gene for B entirely embedded in gene for A, just have different reading frames -complex genome -doesn’t occur in eukaryotes, only phages w/ ssDNA Table 32-2: The Standard Genetic Code (do not need to memorize) o All codons have meaning o UAA, UAG, UGA do not code for an amino acid and are called “nonsense” codons, they serve as termination sequences o The genetic code is unambiguous, each of the 61 “sense” codons encodes for only 1 AA o The genetic code is degenerate. Except for Met and Trp each aa is encoded by more then one codon. These are called synonymous codons. o The genetic code is nearly universal o 1st position on left o 2nd is horizontal o 3rd is on right o start does for Met Codons for the same (or similar) amino acids tend to be similar in sequence: o XYX likely encodes for a hydrophobic aa o XRX likely encodes for a polar or charged aa o GAX encodes for the acidic aa o Y is a pyrimidine o R is a purine Third base degeneracy—(5` residue of the anticodon) o Often the 3rd base in a codon is irrelevant o Eight aa are encoded by the 1st two bases: o Leu: CUX Pro: CCX Arg: CGX Val: GUX Thr: ACX Gly: GGX Ser: UCX Ala: GCX o Seven aa are encoded by a pyrimidine base in the 3rd position o Phe: UUY Asn: AAY Ser: AGY Tyr: UAY Asp: GAY His: CAY Cys: UGY o Five aa and the stop codon are coded by a purine base in the 3rd position: o Leu: UUR Glu: GAR Gln: CAR Arg: AGR Lys: AAR stop: UAR o One aa is coded by any base but G in the 3rd position o Ile: AUX x=U, C, or A o Two aa are coded where the 3rd base must be a G:
  2. 2. o Met: AUG Trp: UGG o The stop codon is coded w/ the requirement that the 3rd base must be an A o Stop: UGA; unlike the other stop codons (UAR) this stop codon is sometimes used to code for selenocysteine, Sec, aka the 21st aa In 1966, Francis Crick proposed the “Wobble hypothesis.”—the 5` base in the anticodon loop is capable of “wobbling” during translation, allowing it to make alternative, non-Watson-Crick H-bonds w/ different codon bases 5` anticodon base pairs w/ 3` codon base C G A U U A or G G U or C I U, C or A 1st base in codon pairs w/ 3rd base in anticodon ribosome forms peptide bond between amino acids
  3. 3. Fig 32-11: Structure of Yeast tRNA Common Features of tRNA o 5` terminal phosphate group o acceptor stem (7 bp, often w/ non WC bp) o D loop (3-4 bp stem, often w/ dihydrouridine, D, in the loop) o Anticodon loop (5 bp stem) o TψC loop (5 bp stem w/ a loop containing pseudouridine, ψ) o Terminal CCA sequence w/ a free 3` OH o The tertiary structure is maintained by H-bonds and stacking interactions o Clover leaf structure o Generally 76 nt in length o Variable loop is most variable part of tRNA o 3-D shape stays pretty much the same Fig 32-12: Tertiary base pairing in yeast tRNA o Typical of long range bps o Interactions stabilize hammer or T-like structure How is the right amino acid matched to its corresponding tRNA? o This is rather important as once an amino-acyl-tRNA is synthesized, the amino acid becomes a “passive participant” in translation o Aminoacyl-tRNA synthetase o Cells have 20 different aminoacyl-tRNA synthetases, one for each aa (except Lys, which in E.coli has 2) o The formation of aminoacyl-tRNAs occurs in 2 steps: 1. the aa is accepted by the synthetase and adenylated 2. the proper tRNA is accepted by the synthetase and the aa is transferred to the 3` terminal residue of the tRNA Overall rxn: There are 2 distinct classes of aminoacyl-tRNA synthetases: Class I nad II o Class I: monomer, first adds the aa to the 2`-OH of the terminal adenylate residue of the tRNA before shifting it to the 3`-OH. Class I enzymes bind to the tRNA acceptor stem helix from the minor groove site o Class II: dimer/tetramer, adds the aa directly to the 3`-OH of the terminal adenylate residue of the tRNA. Class II enzymes bind to the tRNA acceptor stem helix from the major groove site.
  4. 4. Minor groove Class I Class II Arg Ala Major Cys Asp groove Gln Asn Glu Gly Ile His Leu Lys Met Phe Trp Pro Tyr Ser Val Thr How does a synthetase recognize its cognate tRNAs? The recognition occurs via various elements, which include one or more of: o At least one base in the anticodon o One or more of the 3 bps in the acceptor stem o The base at position 73 (unpaired base preceding the CCA end)
  5. 5. For example: E.coli glutaminyl-tRNAGln synthetase uses: o The central U in the CUG anticodon o Contacts from the carboxyl grp of Asp235 to the 2-NH2 grp of G3 (minor groove) of the G3:C70 bp in the acceptor stem o Contacts w/ the “discriminator base” o A mutant synthetase (D235N) will acylate noncognate tRNAs w/ Gln May 26, 2006 Recognition of the Anticodon o Changing the anticodon of either tRNATrp (CCA) or tRNAVal (CAC) to CAU, the anticodon for the Metionine codon AUG, transforms each of the tRNAs into a substrate for methionyl-tRNAMet synthetase o Reversing the Met anticodon of tRNAMet (CAU) to UAC transforms it into a substrate for valy-tRNAVal synthetase o Red dots are being recognized by synthetase o Anticodon CAU finds codon AUG o If you change the anticodon, it has a large effect of what gets put onto tRNA A single acceptor stem bp defines tRNAAla o All tRNAAla, from archaebacteria to eukaryotes, possess the noncanonical G3:U70 bp in the acceptor stem. Changing this to G:C, A:U, or U:G abolishes its ability to be aminoacylated w/ Ala (when base paired w/ Uracil, the 2-NH2 of G3 in the minor groove lacks an H-bonding partner Mutation of a sense codon to a nonsense codon (UAA, UAG, or UGA) results in a truncated polypeptide. However, mutations elsewhere in the genome can suppress the effect of nonsense mutations (nonsense suppression). These suppressions arise from mutations in the tRNA genes that alter the anticodon so that the mutant tRNA read a stop codon and insert an amino acid. For example, changing the anticodon of tRNATyr from GUA to CUA allows tRNA to read the UAG stop codon and insert a Tyr. Suppression tRNAs are generated from minor tRNA species w/in a set of isoacceptor tRNAs, so there is no loss of essential tRNA. UAG: amber stop codon UAA: ochre stop codon UGA: opal stop codon Ribosomes o Ribosomes are large, compact ribonucleoprotein complexes that are found in the cytosols of all cells (also in the mitochondrial matrix and the stroma of chloroplasts). o They are composed of two subunits, each of which contains ribosomal RNAs (rRNAs) and proteins. o In prokaryotes, ribosomes have a sedimentation coefficient of 705 and the subunits are a 305 particle and a 505 particle
  6. 6. o In eukaryotes, the ribosomes have a sedimentation coefficient of 805 and the subunits are a 405 particle and a 605 particle In prokaryotic ribosomes, o there is only one copy of each ribosomal protein per 705 ribosome, except for protein L7/L12, of which there are 4 copies. o Only one protein is common to both the large and small subunits: S20=L26. o The largest ribosomal protein is S1 (557 aa, 61.2 kDa) and the smallest is L34 (46 aa, 5.4kDa). o There is very little sequence homology between ribosomal proteins, although they tend to be rich in Lys and Arg, and have very few aromatic aa residues. o However, comparing ribosomal proteins between different organisms reveals considerable evolutionary conservation of sequence 30S + 50 S  70 S The ribosome has 3 sites for RNA binding: 1. A site (aminoacyl) 2. P site (peptidyl) 3. E site (exit) Mechanics of Traslation There are 3 steps in translation: 1. Initiation 2. elongation 3. termination Each of these stages requires specific proteins to interact w/ the mRNA, tRNA and/or the ribosome. These proteins are the initiation factors (IFs), the elongation facts (EFs) and the release factors (RFs). Initiation: In prokaryotes, the first aa is formylmethionine The same aminoacyl-tRNA synthetase attaches methionine to both tRNAMet and tRNAFMet. However, Met- tRNAMet is not a substrate for formylation by transformylase. fMet-tRNAfMet is recognized by IF-2 whereas Met-tRNAMet is recognized by EF-Tu. E.coli proteins are postranslationally modified by deformylation of the fMet residue.