Chapter 27 Protein Metabolism 27 1 The Genetic Code Ribosomes are the site of protein synthesis from amino acids Aminoacyl tRNAs tRNA Translation tRNAs with an amino acid attached o Aminoacyl tRNA synthetases the overall process of mRNA guided protein synthesis catalyze the addition of an amino acid to The Genetic Code Was Cracked Using Artificial mRNA Templates Reading frame Codon Initiation codon polypeptide in all cells a triplet of nucleotides that codes for a specific amino acid a new codon begins every three nucleotide residues AUG the most common signal for the beginning of a o AUG also codes for methionine Termination codons UAA UAG UGA also called stop codons or nonsense codons o Signal the end of polypeptide synthesis and do not code for any amino acids Open reading frame among 50 or more codons ORF a reading frame without a termination codon o Usually correspond to genes that encode proteins Degenerate an amino acid may be specified by more than one codon Wobble Allows Some tRNAs to Recognize More than One Codon When several different codons specify one amino acid the difference between them usually lies at the third base position Anticodon codons Wobble hypothesis a three base sequence on the tRNA that base pairs with mRNA o The first two bases of an mRNA codon always form strong Watson Crick base pairs with the tRNA anticodon and confer most of the coding specificity o The first base of the anticodon determines the number of codons recognized by the tRNA When the first base is C or A base pairing is specific and only one codon is recognized When the first base is U or G binding is less specific and the tRNA can recognize two different codons When inosine is the first nucleotide three different nucleotides can be recognized o When an amino acid is specified by several different codons the codons that differ in either of the first two bases require different tRNAs o A minimum of 32 tRNAs are required to translate all 61 codons Because the wobble base pairs loosely it permits rapid dissociation of the tRNA from its codon during protein synthesis Translational Frameshifting and RNA Editing Affect How the Code Is Read the addition deletion or alteration of nucleotides in the RNA the reading frame is offset Translational frameshifting RNA editing in a manner that affects the meaning of the transcript adenosine deaminases that act on RNA ADARs APOBEC apoB mRNA editing catalytic peptide family of enzymes o AID activation induced deaminase 27 2 Protein Synthesis Protein Biosynthesis Takes Place in Five Stages Stage 1 Activation of Amino Acids o Two requirements for synthesis of a polypeptide The carboxyl group of each amino acid must be activated to facilitate formation of a peptide bond A link must be established between each new amino acid and the information in the mRNA that encodes it These are met by attaching the amino acid to a tRNA in the first stage of protein synthesis in the cytosol o Amino acids are attached to tRNA using ATP and Mg2 dependent activating enzymes known as aminoacyl tRNA synthetases Stage 2 Initiation o The mRNA bearing the code for the polypeptide binds to the smaller ribosomal subunit and to the initiating aminoacyl tRNA The large ribosomal subunit then binds to form the initiation o Initiation requires GTP and is promoted by cytosolic proteins called complex initiation factors Stage 3 Elongation amino acid units o The polypeptide is lengthened by covalent attachment of successive o Elongation requires elongation factors o The binding of each incoming aminoacyl tRNA and the movement of the ribosome along the mRNA are facilitated by the hydrolysis of GTP Stage 4 Termination and Ribosome Recycling o Release factors aid the release of the polypeptide o Ribosome is recycled Stage 5 Folding and Posttranslational Processing o The polypeptide may undergo enzymatic processing before or after folding Removal of one or more amino acids Addition of acetyl phosphoryl methyl carboxyl or other groups to amino acids Proteolytic cleavage Attachment of oligosaccharides or prosthetic groups The Ribosome Is a Complex Supramolecular Machine The ribosome is a ribozyme Ribosomes of eukaryotic cells are larger and more complex than bacterial ribosomes 3 end Transfer RNAs Have Characteristic Structural Features tRNAs consist of a single strand of RNA folded into a precise three dimensional structure Most tRNAs have a guanylate residue at the 5 end and all have CCA 3 at the Hydrogen bonding pattern of tRNA forms a cloverleaf structure with four Amino acid arm the arm that can carry a specific amino acid esterified by arms its carboxyl group to the 2 or 3 hydroxyl group of the A residue at the 3 end of the tRNA Anticodon arm D arm T C arm contains the unusual nucleotide dihydrouridine contains ribothymidine and psuedouridine the arm that contains the anticodon Stage 1 Aminoacyl tRNA Synthetases Attach the Correct Amino Acids to Their tRNAs Each enzyme is specific for one amino acid The reaction catalyzed by an aminoacyl tRNA occurs in two steps o Am enzyme bound intermediate aminoacyl adenylate is formed o The aminoacyl group is transferred from enzyme bound aminoacyl AMP to its corresponding tRNA o Two high energy phosphate bonds are expended for each amino acid activated rendering the reaction irreversible Proofreading by Aminoacyl tRNA Synthetases o The aminoacylation of tRNA accomplishes two things It activates an amino acid for peptide bond formation It ensures appropriate placement of the amino acid in a growing polypeptide o If available binding interactions do not provide sufficient discrimination between two substrates the necessary specificity can be achieved by substrate specific binding in two successive steps Interaction between an Aminoacyl tRNA Synthetase and a tRNA A Second Genetic Code Stage 2 A Specific Amino Acid Initiates Protein Synthesis All organisms have two tRNAs for methionine o One is used exclusively when 5 AUG is the initiation codon o The other is used to code for a Met residue in an internal position in a polypeptide N formylmethionyl tRNAfMet fMet tRNAfMet is formed in two reactions o Methionine is attached to tRNAfMet by the Met tRNA synthetase o A transformylase transfers a formyl group from N10 formyltetrahydrofolate to the amino group of the Met residue Transformylase is more selective than Met tRNA synthetase The Three Steps of Initiation o Initiation requires The 30S ribosomal subunit The mRNA coding for the polypeptide to be made
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