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Protein Metabolism Chapter 27 Protein Synthesis: an overview Proteins are end products Production must match cellular needs Must be targeted Must be degraded when no longer needed Protein Synthesis is well understood Complex biosynthetic pathway Has been a major emphasis in biochemistry Critical importance in every cell Protein Synthesis: an overview Translation mRNA-directed biosynthesis of polypeptides Requires about 300 macromolecules 70 ribosomal proteins 20+ enzymes to activate amino acid precursors 12+ enzymes for initiation, elongation, termination 100+ enzymes for final processing 40+ kinds of tRNA and rRNA Protein Synthesis: an overview Translation Requires accurate linkage of 20 AA In order specified by mRNA Uses up to 90% of cell biosynthetic energy Occurs at a high rate of speed 100 residue polypeptide takes about 5 sec Must coordinate with targeting and degradation The Genetic Code: 1950’s Where proteins are synthesized: Paul Zamecnik and collegues Determined ribosomes were the site of protein synthesis Activation of amino acids: Mahlon Hoagland and Zamecnik Formation of aminoacyl-tRNAs Catalyzed by aminoacyl-tRNA synthetases The Genetic Code: 1950’s Crick’s adaptor hypothesis Explained how the 4-letter language of nucleic acids was translated into the 20letter language of proteins Proposed that it was a small nucleic acid Part bonds to a specific amino acid part recognizes the codon The Genetic Code: 1960’s Determination of the triplet character Francis Crick and Sidney Brenner: 1961 Induced mutations in bacteriophage T4 Formulated the following hypotheses: The code is read in sequence from a fixed point The code is comma-free The code is a triplet code Codon: group of bases specifying an AA Triplet bases=codon The code is degenerate The Genetic Code: 1960’s Francis Crick and Sidney Brenner: 1961 Used frameshift mutations Observed that insertions or deletions shifted the frame Reading frame: established by the first codon Did not absolutely prove the triplet code The Genetic Code: 1960’s Genes are colinear with their specified polypeptides Charles Yanofskey Used E.coli mutants for tryptophan synthase Specified by the trpA gene Mutations in the gene corresponded to changes in the protein The Genetic Code: 1960’s What were the 3 base codons for each amino acid? The Genetic Code: 1960’s Deciphering the Code Marshall Nirenberg and Heinrich Matthaei: 1961 Revealed the base composition of codons, but not the base sequence The Genetic Code: 1960’s Deciphering the Code Nirenberg and Philip Leder: 1964 Determined about 50 of the 64 possible triplet codons The Genetic Code: 1960’s Deciphering the Code H. Gobind Khorana: 1964 Completed the dictionary Reconfirmed the triplet code Confirmed I.D. of many codons Filled out missing portions of the genetic code 61 of 64 codons specify AA Other 3: termination codons The Genetic Code: 1960’s Meanings for all codons established by 1966 Cracking the genetic code is regarded as one of the most significant scientific discoveries of the twentieth century Nature of the Code The code is DEGENERATE Does not mean that the code is inaccurate or imperfect Nature of the Code The code is DEGENERATE AA may be specified by more than one codon: AA specified by 6 codons: Arg, Leu, Ser AA specified by 4,3 or 2 codons: most AA specified by 1 codon: Methionine (AUG) Tryptophane (UGG) Nature of the Code The code is DEGENERATE Synonyms: codons that specify the same AA Initiation (start) codon: AUG Codons usually differ only in the 3rd base GUG also: or codes for Val AUG also codes for Methionine Stop codons (Nonsense codons): UAA, UAG, UGA Do not code for an AA Nature of the Code The arrangement of the code is nonrandom Nature of the Code The reading frame is correctly set in the beginning of the mRNA readout Initiation Codon: signals start AUG: in prokaryotes and eukaryotes Codes for Met if occurs internally Reading frame moves from one triplet to the next Nature of the Code The reading frame is correctly set in the beginning of the mRNA readout Termination codons (aka: STOP codons; nonsense codons) Signal end of protein synthesis Found first in E. coli as single base mutations Called UAG: amber; UAA: ochre; UGA: opal Eg: an amber mutation changes another codon into UAG Nature of the Code The reading frame is correctly set in the beginning of the mRNA readout Open Reading Frame Sequence of codons (50 or more) without a termination codon Long ones usually correspond to genes for proteins Eg: protein with MW of 60,00=open reading frame of ~500 codons Nature of the Code The reading frame is correctly set in the beginning of the mRNA readout A mistake of a single base will put all subsequent codons out of register Nature of the Code Some viral DNA segments contain overlapping genes in different reading frames Any nucleotide sequence has 3 potential reading frames Nature of the Code Some viral DNA segments contain overlapping genes in different reading frames Any nucleotide sequence has 3 potential reading frames In theory: same sequence could code for 2 or 3 different polypeptides Most DNA sequences: one reading frame; one protein product Nature of the Code Some viral DNA segments contain overlapping genes in different reading frames Frederick Sanger: 1976 DNA of bacteriophage 0X174 Found genes within genes Bacteria also show such coding economy Overlapping genes: in small, single stranded DNA phages Nature of the Code The genetic code is nearly universal A basic concept One organism can translate genes of another Eg: E. coli can translate human genes Basis of genetic engineering Suggests common evolutionary ancestor Single genetic code Strong selection against mutation Nature of the Code The genetic code is nearly universal Minor Variants have been found Mammalian Mitochondria (1981) Contain genes Have variations on the standard genetic code AUA=STOP UGA=Trp (not STOP) AGA, AGG= STOP (not Arg) Increased degeneracy simplifies code Nature of the Code The genetic code is nearly universal Some variants in prokaryotes, protista UGA= STOP and selenocysteine (sometimes) Transfer RNA NA do not specifically bind AA Question: How do cells translate code of Base Sequence into language of polypeptides? Adaptor Hypothesis of Francis Crick Postulated: Genetic code read by molecules that recognize specific codon That molecule carries corresponding AA tRNA was Crick’s adaptor molecule Transfer RNA Primary and Secondary structures Robert Holley: 1965 Reported 1st base sequence of a biologically significant NA Took him 7 yrs Sequenced yeast Alanine tRNA (tRNAAla) Invented methods to sequence RNA Had to purify: yeast tRNAAla has 10 modified bases … made it harder! Transfer RNA Techniques now vastly improved Can sequence in a few days Know sequence for over 300 tRNAs tRNA: primary structure Vary in length 60-95 nucleotides Most are ~75 nucleotides tRNA: secondary structure 5’ terminal phosphate group Acceptor (Amino Acid Stem): 7bp stem Includes 5’ terminal nucleotide May contain non-WC base pairs AA residue: 3’-terminal OH group tRNA: secondary structure D arm (DHU: dihydrouridine arm) 3-4 bp stem Ends in loop Loop often has modified base: dihydrouridine (D) Function? tRNA: secondary structure Anticodon Arm 5 bp stem Ends in loop Loop contains anticodon 3 bases Complimentary to codon tRNA: secondary structure T arm 5bp stem Ends in loop with modified bases Function? Termination All tRNA terminates in CCA And a free 3’-OH group tRNA: secondary structure Variable arm Greatest variability 3-21 nucleotides May have stem up to 7 bp Invariant positions Always same base 13 tRNA: secondary structure Semivarients 8 Always a purine, or always a pyrimidine Mostly in loops Purine on 3’ side of anticodon: modified Correlated invarients Pairs of non-stem nucleotides Are base-paired in all tRNA Tertiary structure Modified tRNA bases Stricking feature of tRNA Up to 20% modified bases Over 50 such bases Functions? tRNA: tertiary structure 3-D structure: 1974 Based on X-ray crystal structure of yeast tRNAPhen Alexander Rich and Sung Hou Kim Aaron Klug (used different crystal form) Molecule assumes L-shaped conformation tRNA: tertiary structure One leg Acceptor and T-stem A-DNA like helix Other leg D-stem Anticodon stem tRNA: tertiary structure Characteristics of L shape Each leg ~60 A long Anticodon and acceptor at opposite ends of molecule ~76A apart 20-25 A wide: essential to function tRNA: tertiary structure Maintained by hydrogen bonds Also stacking interactions Similar to proteins 9 bp cross-link the tertiary structure Mainstay All non W-C base pairs but one Most are invariant, or semivarient Very compact Aminoacyl-tRNA synthetases Group of enzymes Are AA specific Catalyze attachment of correct AA to correct tRNA AA to 3’ terminal of acceptor stem Forms aminoacyl-tRNA 2 classes Based on structure Differ in 2nd reaction step Aminoacyl-tRNA synthetases Reaction energy derived from ATP 2 steps First: activation of AA Formation of enzyme bound intermediate: aminoacyl-AMP Second: formation of aminoacyl-tRNA Aminoacyl group is tranfered to correct tRNA Method depends on enzyme class Aminoacyl-tRNA synthetases Overall reaction: irreversible AA+tRNA+ATP Aminoacyl-tRNA + Amp + PPi Aminoacyl-tRNA synthetases Cells have at least one aminoacyl-tRNA synthetase for each standard AA Diverse group Over 100 characterized Each: 4 subunits Between organisms: same enzyme; considerable sequence homology Between enzymes: little similarity Methods of recognition between enzyme and AA are idiosyncratic Aminoacyl-tRNA synthetases Proofreading: correct AA to correct tRNA Critical Occurs at several sites When AA binds (activation to aminoacyl-AMP) Enzyme has a second active site Will catalyze removal of incorrect AA Based on 3-D structure Third site: sometimes: removes incorrect AA Aminoacyl-tRNA synthetases Proofreading critical Esp with structurally similar AA Second genetic code: Interactions between synthetases and tRNA Critical to accuracy of protein synthesis Complex “coding rules” Some nucleotides are recognition factors Codon-Anticodon Interactions tRNA is selected by codon-anticodon interactions Called Codon recognition Aminoacyl group not involved Variable third position: large part of code degeneracy Isoaccepting tRNAs+different TRNAs specific for same AA Codon-Anticodon Interactions Many tRNAs can bind to multiple codons (specify cognate AA) Non W-C bp often at 3rd position The Wobble Hypothesis: accounts for codon degeneracy Developed by Crick The Wobble Hypothesis First two codon –anticodon pairs are typical W-C: establish structural constraints Third codon position Called the wobble base Allows limited conformational adjustments Non W-C pairs can be accomodated Called wobble pairing The Wobble Hypothesis Crick deduced the most likely wobble pairs C or A: only with W-C pairs U in anticodon: A or G in Codon G in anticodon: U or C in Codon U in anticodon: U, C or A in Codon The Wobble Hypothesis Minimum “set of tRNAs for translation 32 tRNAs 31 to translate 61 coding triplets Initiation requires separate tRNA Most cells have >32 tRNAs If either of first two bases are different on codons for the same AA: require different tRNAs (isoaccepting tRNA) Ribosomes History Albert Claude (late 1930’s) First observed Used dark field microscopy Called them microsomes George Palade (mid 1950’s) EM Established they were not artifacts Ribosomes Name:~2/3 RNA; 1/3 protein Microsome: artifactual vesicle, in EM Ribosome: site of protein synthesis; abundant in cells that make a lot of protein Paul Zamecnik (1955) Confirmed ribosomes were the site of protein synthesis Ribosomes Ribosomal protein synthesis: 3 phases Chain initiation Chain elongation Chain termination Ribosomes Structure of prokaryote ribosome Spheroid particle 70S ~250A dia Two subunits (discovered by James Watson) Small: 30s 16s rRNA molecule 21 different polypeptides Large: 50S 5S rRNA 23S rRNA 32 different polypeptides Ribosomes Structure of prokaryote ribosome ~20,000 ribosomes in E. coli ~ 80% of cell RNA ~ 10% of cell protein Ribosomes Have been crystallized recently 3-D studies: Image reconstruction by EM Aaron Klug Used images from several views Mathematical reconstruction Small subunit “Mitten”- shaped Head, base, cleft, platform Ribosomes 3-D studies: continued Large subunit Spheroidal with 3 protuberances Central protuberance Ridge Stalk Tunnel ~25A dia 100-120 A long Originates in a cleft between protuberances Ribosomes Secondary structure of rRNA “flower-like” in the 16S rRNA of 30S subunit 4 domains: sequenced (1542 nucleotides) 46% base paired Double-helical areas tend to be short ~8bp Shape largely determines shape of 30S subunit Large subunit: rRNA has been sequenced 5S: 120 nucleotides 23S: 2904 nucleotides Extensive secondary structure Ribosomes Ribosomal Proteins Insoluble: difficult to isolate Naming conventions From small subunit: S From large subunit: L Followed by number based on electrophoretogram: Eg: S20/L26 All 52 E. coli proteins have been sequenced Little sequence similarity Most are rich in Lys, Arg Ribosomes Ribosomal subunits are self-assembling Ribosomal architecture: Component positions have been determined by Immune EM Verified: eg: neutron diffraction measurement Functional Components Identified by affinity labeling techniques Ribosomes Ribosomal architecture: Functional Components Binding mRNA on small subunit Anticodon binding site 3’ end of 16S rRNA Located on “platform” Binds from “head” to “base” Small subunit cleft GTP reactions Large subunit Stalk (4 L7/L12 subunits) Ribosomes Ribosomal architecture: Functional Components: continued Peptidyl transferase function Ribosome binding to membranes Large subunit Adjacent to exit tunnel Large subunit Catalyzes peptide bond formation “valley” of small subunit Main job: biochemical tasks Also, tRNA binding Small subunit: binds mRNA and tRNA Ribosomes Eukaryotic Ribosomes Resemble prokaryotic Larger, more complex ~23 nm dia Sedimentation coefficient= ~80S 2 subunits Large subunit: 60S Small subunit: 40S Ribosomes Eukaryotic Ribosomes Large subunit rRNA: 3 5S 5.8S 28S 49 polypeptides Small subunit rRna: 18S 33 polypeptides