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1 You should be able to explain the above from this lecture. 2 `Three kinds of RNA molecules perform different but cooperative functions in protein synthesis: 1. Messenger RNA (mRNA) carries the genetic information copied from DNA in the form of a series of three-base or triplet code (as we shall see) each of which specifies a particular amino acid. 2. Transfer RNA (tRNA) is the molecular adaptor that deciphers the triplet code in mRNA. Each type of amino acid has its own type of tRNA, which binds it and escorts it to the growing end of a polypeptide chain if called for by the next triplet on the mRNA. The correct tRNA with its attached amino acid is selected based on it having a three-base sequence that can base-pair with its complementary triplet in the mRNA. 3. Ribosomal RNA (rRNA) associates with a set of ribosomal proteins to form ribosomes. These large macromolecular complexes catalyze the polymerization of amino acids into protein chains. They also bind tRNAs and various accessory molecules necessary for protein translation. Here we consider tRNA 3 During translation, polypeptide chains are formed by covalently linking the carboxyl group of one amino acid to the amino group of the next. However, as you heard from Professor Kahne, the reaction, is energetically unfavorable, with unbonded amino acids being favored by about 2.4 kcal/mol. This means that the polypeptide chains that make up proteins are thermodynamically unstable in water, however, as you already heard the spontaneous hydrolysis of peptide bonds is extremely slow at physiological pH. Synthesis of peptide bonds therefore poses a thermodynamic challenge to the cell because of the free energy cost. As we have seen before, the cell often solves this kind of problem by coupling the unfavorable reaction to favorable reactions so that the net change in free energy is negative, allowing the overall reaction to proceed. In this case, ATP hydrolysis is used to create a high energy bond between a tRNA and the appropriate amino acid in a process called “tRNA charging.” Before the codon specifying a given amino acid can be recognized by a particular tRNA, the appropriate amino acid must be covalently coupled to that tRNA. This essential process is catalyzed by an enzyme called aminoacyl-tRNA synthetase. Each of the 20 different synthetases recognizes one amino acid and all its compatible tRNAs. These coupling enzymes link an amino acid to the free 3’ hydroxyl of the adenosine at the 3’ terminus of tRNA molecules. ATP hydrolysis is required for this 4 reaction to occur, and the amino acid is linked to the tRNA by a high-energy bond. The term, high-energy bond, refers to the fact that the breaking of this bond is favorable and has a large and negative ∆G0rxnThe energy of the bond between the amino acid and the tRNA subsequently drives the formation of bonds between adjacent amino acids in a growing protein chain, overcoming the energetic cost of forming a peptide bond. Charged tRNAs are available to be selected on the ribosome (as we will come to) by pairing between the anticodon with the corresponding codon in mRNA. 4 7 8 9 10 11 To ensure proper protein function the correct sequence of amino acids must be linked together during the course of translation. The first step towards ensuring translational accuracy depends on the tRNA synthetase which is responsible for linking the correct amino acid to each tRNA. Most synthetases select the correct amino acid by a two-step mechanism that involves two discrete sites on the enzyme. First, the correct amino acid has the highest binding affinity for the active-site pocket (synthesis site) of its synthetase and is therefore favored over the other 19. Amino acids that are larger than the correct one are excluded from the active site based on size. However, this mechanism for proof-reading the candidate amino acid is not sufficient since distinguishing two amino acids of similar size is not possible at this site. For example, isoleucine and valine differ by only a single methyl group and both are likely to fit into the synthesis site of the synthetase meant to only load isoleucine on the appropriate tRNA. Fortunately, a second proof-reading step occurs after the candidate amino acid has associated with the synthetase and the tRNA. In this step the candidate amino acid is shifted into a second pocket in the synthetase. The precise dimensions and shape of this second site excludes the correct amino acid but allows access by closely related amino acids. Once an amino acid enters this editing site, it is hydrolyzed from the tRNA and released from the enzyme. This hydrolytic editing is analogous to the editing by DNA polymerases and increases the overall accuracy of tRNA charging. 12 The tRNA synthetase also recognizes the correct set of tRNAs based on their structural and chemical characteristics. Most tRNA synthetases directly recognize the matching tRNA anticodon while others recognize the nucleotide sequence of the 3’ stem. Thus, several specialized sites on the synthetase will recognize and bind nucleotides at several positions on the tRNA. 12 We have just seen how tRNAs are charged with the appropriate amino acid. We also know that the anticodon of the tRNA reads the codon sequence of the mRNA, which in turn determines the sequence of amino acids linked together during protein synthesis. If the many components that participate in translation (mRNA, aminoacyl-tRNAs) had to depend on random collisions in solution, the frequency would be so low that amino acid polymerization would be very inefficient. The efficiency of translation is greatly increased by a remarkable macromolecular machine made up of RNA and protein, called the ribosome. The ribosome is able to catalyze the polymerization of a protein chain at the rate of up to five amino acids per second. Small proteins of 100 – 200 amino acids are therefore made in a minute or less. 13 The ribosome consists of small and large subunits which are composed of one or two RNA molecules, respectively, and numerous proteins. When not directly participating in translation, the two ribosomal subunits exist as separate entities in the cytosol. They only come together when translation is initiated (usually close to the 5’ end of the mRNA). The small subunit facilitates base pairing between the codon and anticodon sequences while the large subunit catalyzes the formation of peptide bonds between amino acids. The RNA molecules, known as ribosomal RNAs, are transcribed from their corresponding non-protein coding genes in the chromosome. The ribosomal proteins are translated from mRNAs copied from their protein coding genes. 14 Each ribosome contains three sites that are able to bind tRNA in conjunction with the mRNA. The designation of each site is based on the type of tRNA that it associates with. The A site binds to the incoming aminoacyl tRNA and is the initial site of interaction between the ribosome and the charged tRNA. The P site binds to the peptidyl tRNA, which is bonded to the growing polypeptide chain. The E site binds to the exiting tRNA that is no longer bonded to the polypeptide chain. Next, we will see how the three sites each contribute to the translation cycle required to add an amino acid to the carboxyl-terminus of the growing polypeptide chain. 15 The mRNA is translated in a 5’ to 3’ direction (codons are oriented 5’XXX-3’) and the protein is synthesized in an amino terminal to carboxy terminal direction 16 17 The addition of each successive amino acid to a growing polypeptide chain takes place in three steps that involve the ribosome, mRNA, tRNA, and several other proteins. Lets begin with a simplified overview of the mRNA translation cycle. Here we join the process with the P site of the ribosome already containing a tRNA bonded to the end of the growing polypeptide of three amino acids. In step 1, a tRNA carrying the next amino acid (tyrosine) in the chain binds to the A site by base pairing with the mRNA codon positioned there. Charged tRNAs rapidly diffuse in and out of the A site but only one for which there is a proper codon/anticodon is selected. 18 19 In step 2, the carboxyl group at the end of the polypeptide chain (in the cartoon, just a methionine) attached to the tRNA in the P site is transferred to the free amino group of the amino acid (tryosine) of the amino acyl tRNA, forming a peptide bond between amino acids methionine and tyrosine. This is the central reaction of translation and is catalyzed by the large ribosomal subunit. As a consequence the growing polypeptide chain is present (momentarily) in the A site and the peptyidyl tRNA in the P site is now uncharged. 21 The peptide bond is formed between the free amino group of the amino acid in the A site and the carboxyl group at the end of the polypeptide chain in the P site. The reaction is driven by the high energy of the acyl linkage between the carboxyl group and the 3’ hydroxyl at the end of the tRNA and is catalyzed by the large subunit of the ribosome. Thus, the energy for peptide bond formation is derived from the expenditure of a molecule of ATP in the charging of tRNA. 22 23 In step 3, the ribosome shifts relative to the mRNA one codon on the 3’ direction. As a consequence, the peptidyl tRNA is moved into the P site and the now uncharged tRNA that had been in the P site is transferred to the E site and released from the ribosome. The shift is catalyzed by a protein called EF-G and involves the hydrolysis of a molecule of GTP as an energy source. 24 25 26 27 28 29 30 This effectively resets the ribosome so it is ready to receive the next amino acyl tRNA in the now vacant A-site and Step 1 is then repeated with a new incoming aminoacyl tRNA. 31 32 The genetic code is a triplet code, with every three nucleotides being decoded from a specified starting point in the mRNA and in the 5’ to 3’ direction. Each triplet is called a codon. Since there are 61 codons for 20 amino acids, it follows that many amino acids being specified by more than one codon. Indeed, only two — methionine and tryptophan — have a single codon; at the other extreme, leucine, serine, and arginine are each specified by six different codons. The different codons for a given amino acid are said to be synonymous. The code itself can be termed degenerate since it contains redundancies. 33 34 When considering how a sequence of triplet codons can be read to determine the sequence of a linear chain of amino acids, it is important to remember that the genetic code does not have inserted punctuation. In other words, once the first codon position has been defined, all of the other codons are defined in a contiguous and continuous sequence with no breaks or interruptions. The phasing of the codons defined by the first is known as the reading frame of that particular mRNA transcript. As shown above, this also means that a particular mRNA theoretically could be translated in three different reading frames because each reading frame is determined by the starting nucleotide of the first codon. The different reading frames define different sequences of codons which will yield different amino acid sequences. Here is where the start codon AUG comes it. It specifies the correct reading frame. In eukaryotes, the start codon is the first AUG, which specifies methionine, downstream of the CAP. AUG can also specify methionine within a reading frame but it only sets the reading frame if it is the first AUG downstream of CAP. 35 36 37 38