Mary K. Campbell Shawn O. Farrell �������������������������� Chapter Twelve Protein Synthesis: Translation of the Genetic Message Paul D. Adams • University of Arkansas 1 Translating the Genetic Message • Protein biosynthesis is a complex process requiring ribosomes, mRNA, tRNA, and mRNA, tRNA, and protein factors • Several steps are involved • Before being • Before being incorporated into growing protein chain, a.a. must be activated by tRNA and aminoacyl-tRNA synthetases 2
The Genetic Code • Salient features of the genetic code • triplet: triplet: a sequence of three bases (a codon) is needed to specify one amino acid needed to specify one amino acid • nonoverlapping: nonoverlapping: no bases are shared between consecutive codons • commaless: commaless: no intervening bases between codons • degenerate: degenerate: more than one triplet can code for the same amino acid; Leu, Ser, and Arg, for example, are each coded for by six triplets • universal: universal: the same in viruses, prokaryotes, and eukaryotes; the only exceptions are some codons in mitochondria 3 The Genetic Code (Cont’d) • The ribosome moves along the mRNA three bases at a time rather bases at a time rather than one or two at a time • Theoretically possible genetic codes are genetic codes are shown in figure 12.2 4
The Genetic Code (Cont’d) • All 64 codons have assigned meanings • 61 code for amino acids • 3 (UAA, UAG, and UGA) serve as termination signals • only Trp and Met have one codon each • the third base is irrelevant for Leu, Val, Ser, Pro, Thr, Ala, Gly, and Arg • the second base is important for the type of amino acid; for example, if the second base is U, the amino acids coded for are hydrophobic acids coded for are hydrophobic • for the 15 amino acids coded for by 2, 3, or 4 triplets, it is only the third letter of the codon that varies. Gly, for example, is coded for by GGA, GGG, GGC, and GGU 5 The Genetic Code (Cont’d) 6
7 The Genetic Code (Cont’d) • Assignments of triplets in genetic code based on several different experiments • synthetic mRNA: • synthetic mRNA: synthetic mRNA: if mRNA is polyU, polyPhe is synthetic mRNA: if mRNA is polyU, polyPhe is formed; if mRNA is poly --- ACACACACACACACACACACA ---, poly( Thr-His ) is formed • binding assay: binding assay: aminoacyl-tRNAs bind to ribosomes in the presence of trinucleotides • synthesize trinucleotides by chemical means synthesize trinucleotides by chemical means • carry out a binding assay for each type of trinucleotide • aminoacyl-tRNAs are tested for their ability to bind in the presence of a given trinucleotide 8
9 tRNA Structure – simple 10 https://wikispaces.psu.edu/display/Bio110nk/From+Gene+to+Protein#FromGenetoProtein-GeneToProtein%3ATheCentralDogma
The Filter-Binding Assay Helps Elucidate the Genetic Code 11 Wobble Base Pairing • Some tRNAs bond to one codon exclusively, but many tRNAs can recognize many tRNAs can recognize more than one codon because of variations in allowed patterns of hydrogen bonding • the variation is called “wobble” “wobble” • wobble is in the first base of the anticodon 12
Base Pairing Combination in the Wobble Scheme 3’ 13 Wobble Base Pairing Alternatives 14
Wobble Base Pairing Hypothesis • The wobble hypothesis provides insight into some aspects of the degeneracy of the code • in many cases, the degenerate codons for a given • in many cases, the degenerate codons for a given amino acid differ only in the third base; therefore fewer different tRNAs are needed because a given tRNA can base-pair with several codons • the existence of wobble minimizes the damage that can be caused by a misreading of the code; for example, if the Leu codon CUU were misread CUC or example, if the Leu codon CUU were misread CUC or CUA or CUG during transcription of mRNA, the codon would still be translated as Leu during protein synthesis 15 Amino Acid Activation • Amino acid activation and formation of the aminoacyl-tRNA take place in two separate steps aminoacyl-tRNA take place in two separate steps • Both catalyzed by amionacyl-tRNA synthetase • Free energy of hydrolysis of ATP provides energy for bond formation 16
Amino Acid Activation (Cont’d) • This two-stage reaction allows selectivity at two levels • the amino acid: • the amino acid: the amino acid: the aminoacyl-AMP remains bound the amino acid: the aminoacyl-AMP remains bound to the enzyme and binding of the correct amino acid is verified by an editing site in the tRNA synthetase • tRNA tRNA: there are specific binding sites on tRNAs that are recognized by aminoacyl-tRNA synthetases. 17 Amino Acid Activation (Cont’d) 18
19 https://wikispaces.psu.edu/display/Bio110nk/From+Gene+to+Protein#FromGenetoProtein-GeneToProtein%3ATheCentralDogma 20
tRNA Tertiary Structure • There are several recognition sites for various amino acids on the tRNA 21 The Ribosome - summary 22 https://wikispaces.psu.edu/display/Bio110nk/From+Gene+to+Protein#FromGenetoProtein-GeneToProtein%3ATheCentralDogma
Chain Initiation • In all organisms, synthesis of polypeptide chain starts at the N-terminal end, and grows from N- terminus to C-terminus terminus to C-terminus • Initiation requires: • tRNA fmet • initiation codon (AUG) of mRNA • 30S ribosomal subunit • 50S ribosomal subunit • 50S ribosomal subunit • initiation factors IF-1, IF-2, and IF-3 • GTP, Mg 2+ • Forms the initiation complex 23 The Initiation Complex 24
The Initiation Complex - simplified 25 https://wikispaces.psu.edu/display/Bio110nk/From+Gene+to+Protein#FromGenetoProtein-GeneToProtein%3ATheCentralDogma Chain Initiation • tRNA met and tRNA fmet contain the triplet 3’-UAC-5’ • Triplet base pairs with 5’-AUG-3’ in mRNA • 3’-UAC-5’ triplet on tRNA fmet recognizes the AUG triplet (the start signal) when it occurs at the beginning of the mRNA sequence that directs polypeptide synthesis • 3’-UAC-5’ triplet on tRNA met recognizes the AUG triplet when it is found in an internal position in the mRNA sequence • Start signal is preceded by a Shine-Dalgarno purine- rich leader segment, 5’-GGAGGU-3’, which usually lies about 10 nucleotides upstream of the AUG start signal and acts as a ribosomal binding site 26
Chain Elongation • Uses three binding sites for tRNA present on the 50S subunit of the 70S ribosome: P (peptidyl) site, A (aminoacyl) site, E (exit) site. (aminoacyl) site, E (exit) site. • Requires • 70S ribosome • codons of mRNA • aminoacyl-tRNAs • elongation factors EF-Tu ( Elongation factor • elongation factors EF-Tu ( Elongation factor temperature-unstable ), EF-Ts ( Elongation factor temperature-stable ), and EF-G ( Elongation factor- GTP ) • GTP, and Mg 2+ 27 Shine-Dalgarno Sequence Recognized by E. Coli Ribosomes 28
Elongation Steps • Step 1 • an aminoacyl-tRNA is bound to the A site • the P site is already occupied • 2nd amino acid bound to 70S initiation complex. Defined by the mRNA • Step 2 • EF-Tu is released in a reaction requiring EF-Ts • Step 3 • the peptide bond is formed, the P site is uncharged • the peptide bond is formed, the P site is uncharged • Step 4 • the uncharged tRNA is released • the peptidyl-tRNA is translocated to the P site • EF-G and GTP are required • the next aminoacyl-tRNA occupies the empty A site 29 Chain Elongation 30
Peptide bond formation 31 Chain Termination • Chain termination requires • stop codons (UAA, UAG, or UGA) of mRNA • RF-1 ( Release factor-1 ) which binds to UAA and • RF-1 ( Release factor-1 ) which binds to UAA and UAG or RF-2 ( Release factor-2 ) which binds to UAA and UGA • RF-3 which does not bind to any termination codon, but facilitates the binding of RF-1 and RF-2 • GTP which is bound to RF-3 • The entire complex dissociates setting free the completed polypeptide, the release factors, tRNA, mRNA, and the 30S and 50S ribosomal subunits 32
Chain Termination 33 Translation – overview 34 https://wikispaces.psu.edu/display/Bio110nk/From+Gene+to+Protein#FromGenetoProtein-GeneToProtein%3ATheCentralDogma
Translation – simple summary tRNA with amino acid attached 3’ 3’ 5’ 35 https://wikispaces.psu.edu/display/Bio110nk/From+Gene+to+Protein#FromGenetoProtein-GeneToProtein%3ATheCentralDogma Components of Protein Synthesis 36
Protein Synthesis • In prokaryotes, translation begins very soon after mRNA transcription • It is possible to have several molecules of RNA • It is possible to have several molecules of RNA polymerase bound to a single DNA gene, each in a different stage of transcription • It is also possible to have several ribosomes bound to a single mRNA, each in a different stage of translation • Polysome: • Polysome: Polysome: mRNA bound to several ribosomes Polysome: mRNA bound to several ribosomes • Coupled translation: Coupled translation: the process in which a prokaryotic gene is being simultaneously transcribed and translated 37 Simultaneous Protein Synthesis on Polysomes • A single mRNA molecule is translated by several ribosomes simultaneously • Each ribosome produces a copy of the polypeptide chain specified by the mRNA • When protein has been completed, the ribosome dissociates into subunits that are used again in dissociates into subunits that are used again in protein synthesis 38
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