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Translation Tjian Lec 26 Protein Translation The genetic code Protein synthesis Machinery Ribosomes, tRNA’s Universal translation: Protein Synthesis Chemical Letters DNA A,T,G,C mRNA A,U,G,C * A,C,D,E,F,G,H, protein I,K,L,M,N,P,Q, R,S,T,V,W,Y *ribosome 20 synthetases ~45 tRNAs (E. coli) 1 tRNA <-> 1 amino acid Amino Acid activation. The two-step process in which an amino acid (with its side chain denoted by R) is activated for protein synthesis by an aminoacyl-tRNA synthetase enzyme is shown. As indicated, the energy of ATP hydrolysis is used to attach each amino acid to its tRNA molecule in a high-energy linkage. The amino acid is first activated through the linkage of its carboxyl group directly to an AMP moiety, forming and adenylated amino acid; the linkage of the AMP, normally an unfavorable reaction, is driven by the hydrolysis of the ATP molecule that donates the AMP. Without leaving the synthetase enzyme, the AMP-linked carboxyl group on the amino acid is then transferred to a hydroxyl group on the sugar at the 3’ end of the tRNA molecule. This transfer joins the amino acid by an activated ester linkage to the tRNA and forms the final aminoacyl-tRNA molecule. The synthetase enzyme is not shown in this diagram. The genetic code is translated by means of two adaptors that act one after another. The first adaptor is the aminoacyl-tRNA synthetase, which couples a particular amino acid to its corresponding tRNA molecule itself, whose anticodon forms base pairs with the appropriate codon on the mRNA. An error in either step would cause the wrong amino acid to be incorporated into a protein chain. In the sequence of events shown, the amino acid tryptophan (Trp) is selected by the codon UGG on the mRNA. Molecular models of valine and isoleucine. The extra methylene group in isoleucine is marked. The synthetases specific for these amino acids are highly discerning. Glutaminyl-tRNA synthetase complex. The structure of this complex reveals that the synthetase interacts with base pair G10:C25 in addition to the acceptor stem and anticodon loop. A comparison of the structure of procaryotic and eucaryotic ribosomes. Ribosomal components are commonly designated by the their “S values,” which refer to their rate of sedimentation in an ultracentrifuge. Despite the differences in the number and size of their rRNA and protein components, both procaryotic and eucaryotic ribosomes have nearly the same structure and they function similarly. Although the 18S and 28S rRNAs of the eucaryotic ribosome contain many extra nucleotides not present in their bacterial counterparts, these nucleotides are present as multiple insertions that form extra domains and leave the basic structure of each rRNA largely unchanged. 30S 50S Head CP L11 A site tRNA Body T.th. 70S H69 h44 5.5 Å Yusupov et al. (2001) Science 292, 883. Formation of the initiation complex. The complex forms in three steps at the expense of the hydrolysis of GTP to GDP and Pi. IF-1, IF- 2, and IF-3 are initiation factors. P designates the peptidyl site, A the aminoacyl site, and E, the exit site. Here the anticodon of the tRNA is oriented 3’ to 5’, left to right. Major Points 1. Alternative RNA splicing: one mechanism evolved to expand diversity of gene products without increasing gene number 2. Control of alternative splicing by positive and negative splicing factors ( analogous to transcriptional activators and repressors) 3. Complexity of organisms reflected by exon numbers and differences between prokaryotic and eukaryotic mRNA’s 4. Universal triplet codon converts NA seq into amino acid seq: total of 64 codes- AUG for Met and START, UAG,UAA&UGA for STOP and the rest for the remaining 19 amino acids 5. Degeneracy of the code, ribosome entry site seq (Shine-Delgarno) anti-codon seq in tRNA 6. High energy, amino-acyl-tRNA’s and specific synthetases 7. Importance of adaptor molecules (ie. tRNA synthetases and anti-codon loop of tRNA’s) 8. Complex structure of ribosomes: protein and RNA components Formation of the initiation complex. The complex forms in three steps at the expense of the hydrolysis of GTP to GDP and Pi. IF-1, IF-2, and IF-3 are initiation factors. P designates the peptidyl site, A the aminoacyl site, and E, the exit site. Here the anticodon of the tRNA is oriented 3’ to 5’, left to right. Elongation cycle : binding of aminoacyl-tRNA, peptide-bond formation, and translocation. First step in elongation (bacteria): binding of the second aminoacyl-tRNA. The second aminoacyl-tRNA enters the A site of the ribosome bound to EF-Tu (shown here as Tu), which also contains GTP. Binding of the second aminoacyl-tRNA to the A site is accompanied by hydrolysis of the GTP to GDP and Pi and releaseof the EF-Tu•GDP complex from the ribosome. The bound GDP is released when the EF-Tu•GDP complex binds to EF-Ts, and EF-Ts is subsequently released when another molecule of GTP binds to EF-Tu. This recycles EF-Tu and makes it available to repeat the cycle. Second step in elongation (bacteria): formation of the first peptide bond. The peptidyl transferase catalyzing this reaction is probably the 23S rRNA ribozyme. The N- formylmthionyl group is transferred to the amino group of the second aminoacyl-tRNA in the A site, forming a dipeptidyl-tRNA. At this stage, both tRNAs bound to the ribosome shift position in the 50S subunit to take up a hybrid binding state. The uncharged tRNA shifts so that its 3’ and 5’ ends are in the E site. Similarly, the 3’ and 5’ ends of the peptidyl tRNA shift to the P site. The anticodons remain in the A and P sites. Third step in elongation (bacteria): translocation. The ribosome moves one codon toward the 3’ end of mRNA, using energy provided by hydrolysis of GTP bound to EF-G (translocase). The dipeptidyl-tRNA is now entirely in the P site, leaving the A site open of the incoming (third) aminoacyl-tRNA. The uncharged tRNA dissociates from the E site, and the elongation cycle begins again. Termination of protein synthesis in bacteria. Termination occurs in response to a termination codon in the A site. First, a release factor (RF1 or RF2 depending on which termination codon is present) binds to the A site. This leads to hydrolysis of the ester linkage between the nascent polypeptide and the tRNA in the P site and release of the completed polypetide. Finally, the mRNA, deacylated tRNA, and release factor leave the ribosome, and the ribosome dissociates into its 30S and 50S subunits. A polyribosome. (A) Schematic drawing showing how a series of ribosomes can simultaneously translate the same eucaryotic mRNA molecule. (B) Electron micrograph of a polyribosome from a eucaryotic cell. Ribosomes and endoplasmic reticulum. The electron micrograph and schematic drawing of a portion of a pancreatic cell show ribosomes attached to the outer (cytosolic) face of the endoplasmic reticulum (ER). The ribosomes are the numerous small dots bordering the parallel layers of membranes. Major Points 1. Multi-step protein synthesis : Initiation - small ribosome subunit loads onto mRNA at the entry site and (AUG) START codon along with IF1,2&3 followed by loading large subunit 2. Three distinct binding pockets/sites in the ribosome complex: P- peptidyl ; A- aminoacyl ; and E - Exit sites 3. Several steps during protein synthesis require hydrolysis of GTP to GDP & Pi (initiation complex formation; amino-acyl-tRNA binding and translocation) 4. Peptidyl transferase reaction is catalyzed by ribozyme (RNA enzyme) within the small ribosomal subunit (23S or 30S) • Eukaryotic protein synthesis involves polyribosomes and a RNP complex in which the 5’ Cap of mRNA is linked to the 3’ poly A via various translation factors .
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