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Chapter 14 Translation

Chapter 14 Translation. MESSENGER RNA Polypeptide chains are specified by open reading frames An open reading frame (ORF) is a contiguous, non-overlapping string of codons.

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Chapter 14 Translation

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  1. Chapter 14 Translation

  2. MESSENGER RNA Polypeptide chains are specified by open reading frames An open reading frame (ORF) is a contiguous, non-overlapping string of codons. Start codon: the first codon of an ORF. It specifies the first amino acid to be incorporated. It defines the reading frame for all subsequent codons. Stop codon: the last codon of an ORF. Polycistronic mRNAs: mRNAs that contain multiple ORFs. Monocistronic mRNAs: mRNAs that contain a single ORF.

  3. Prokaryotic mRNAs have a ribosome binding site that recruits the translational machinery RBS (ribosome binding site): a short sequence upstream of the start codon that facilitates binding of a ribosome. AGGAGG. Also referred to as a Shine-Dalgarno sequence. Interacts with 16S rRNA In case that the start codon of downstream ORF overlaps the stop codon of upstream ORF, for example, with AUGA, translation of two ORFs is linked. This is known as translational coupling.

  4. Eukaryotic mRNAs are modified at their 5’ and 3’ ends to facilitate translation The 5’ cap is a methylated guanine nucleotide at 5’ end of mRNA. Recruits the ribosome. The ribosome moves in a 5’  3’ direction until it encounters an AUG in a process called scanning. The Kozak sequence (PuNNAUGG) interacts with initiator tRNA. Poly-A tail promotes efficient recycling of ribosomes

  5. TRANSFER RNA tRNAs are adaptors between codons and amino acids tRNAs are between 75 and 95 nt in length. 1. All tRNAs end at the 3’ terminus with the sequence 5’-CCA-3’. 2. Several unusual bases are present in the primary structure of tRNAs. ΨU (Pseudouridine), D (dihydrouridine), hypoxanthine, thymine, methylguanine -----probably improves tRNA function

  6. tRNAs share a common secondary structure that resembles a cloverleaf The acceptor stem is formed by pairing between the 5’ and 3’ ends of the mRNA molecules. It is the site of amino acid attachment. The ΨU loop. The unusual base ΨU is often found within the sequence 5’-TΨUCG-3’. The D loop. The anticodon loop. The anticodon is always bracketed on the 3’ end by a purine and on its 5’ end by uracil. The variable loop. It varies in size from 3 to 21 bases.

  7. tRNAs have an L-shaped three-dimensional structure X-ray crystallography reveals an L-shaped tertiary structure. The terminus of the acceptor stem is about 70 Å away from the anticodon loop at the other end. Three kinds of interactions stabilize this structure: 1. The formation of the two extended regions of base pairing results in additional base stacking interactions. 2. unconventional hydrogen bonds between bases in different helical regions. 3. Interactions between the bases and the sugar-phosphate backbone.

  8. ATTACHMENT OF AMINO ACIDS TO tRNA tRNAs are charged by the attachment of an amino acid to the 3’ terminal adenosine nucleotide via a high-energy acyl linkage The carboxyl group of an amino acid is linked to 2’ or 3’-OH of the terminal adenosine nucleotide through the high energy acyl linkage. The energy released when the bond is broken drives the peptide bond formation. Aminoacyl-tRNA synthetases charge tRNAs in two steps 1. adenylylation – transfer of AMP 2. tRNA charging Class I tRNA synthetases attach a.a. to 2’-OH and are generally monomeric. Class II enzymes attach a.a. to 3’-OH and are typically dimeric or tetrameric. The amino acid rapidly equilibrates between attachment at 3’ and 2’-OH

  9. Each aminoacyl-tRNA synthetase attaches a single amino acid to one or more tRNAs Most organisms have 20 different tRNA synthetases. An exception in some bacteria: amination of Glu-tRNAGln to Gln-tRNAGln

  10. tRNA synthetases recognize unique structural features of cognate tRNAs Specificity determinants: the acceptor stem and the anticodon loop Discriminator in the acceptor stem: Changing a particular base pair in the acceptor stem converts the recognition specificity of a tRNA from one synthetase to another. The anticodon loop, including recognition of the anticodon itself, contributes to discrimination. However, in case of serine, AGC and UCA are completely different from each other. The specificity determinants should be outside of the anticodon. The set of tRNA determinants that enable synthetases to discriminate among tRNAs is referred to as the second genetic code.

  11. Aminoacyl-tRNA formation is very accurate Seletion of the correct amino acid is more difficult. Nevertheless, less than 1 in 1,000 tRNAs is charged with the incorrect amino acid. Tyrosine vs. phenylalanine -OH allows the synthetase to discriminate between the two amino acids. Valine vs. isoleucine -CH3 leads to 100 fold difference, which is unacceptable.

  12. Some aminoacyl tRNA synthetases use an editing pocket to charge tRNAs with high accuracy In addition to the catalytic pocket for adenylyation, isoleucyl tRNA synthetase has a nearby editing pocket for proofreading the product of adenylylation reaction. The pocket functions as a molecular sieve. AMP-isoleucine cannot enter the pocket whereas AMP-valine can fit into the pocket and is hydrolyzed.

  13. The ribosome is unable to discriminate between correctly and incorrectly charged tRNAs A mutant tRNA with a nucleotide substitution in the anticodon delivers its usual cognate amino acid to the wrong codon. Cysteinyl-tRNACys can be converted to alanine-tRNACysby chemical reduction. This reduction results in introduction of alanines at the cysteine codons. High fidelity of tRNA synthetases are required for accurate decoding of mRNAs.

  14. THE RIBOSOME The ribosome is the macromolecular machine that directs the synthesis of proteins. More complex than the minimal replication and transcription machinery. The ribosome is composed of at least 3 RNAs and more than 50 proteins, with a molecular mass of greater than 2.5 megadaltons. The speed of translation is 20 amino acids (60 nt) per second in prokaryotes. Similar to the rate of RNA transcription 2-4 amino acids per second in eukaryotes

  15. Transcription and translation are coupled in prokaryotes.

  16. The ribosome is composed of a large and a small subunit Peptidyl transferase center: a part of the large subunit that is responsible for the formation of peptide bonds. Decoding center: a part of the small subunit where charged tRNA decode the codon units of the mRNA. 30S+50S=70S 40S+60S=80S 2/3 RNA + 1/3 protein

  17. The large and Small subunits undergo association and dissociation during each cycle of Translation The ribosome cycle: the sequence of association and dissociation of the ribosome.

  18. Polyribosome (or polysome): an mRNA bearing multiple ribosomes A single ribosome contacts with 30 nt, but mRNA can bind one ribosome for every 80 nucleotides. New Amino acids are attached to the C-terminus of the growing polypeptide chain Polypeptides are synthesized in the N- to C-terminal direction.

  19. Peptide bonds are formed by transfer of the growing polypeptide chain from one tRNA to another The ribosome catalyzes the formation of a peptide bond between the amino acids attached to tRNAs. Two consequences of the peptidyl transferase reaction: 1. The N-terminus of the protein is synthesized before the C-terminus. 2.The growing polypeptide chain is transferred from the peptidyl-tRNA to the aminoacyl-tRNA. No ATP is required, but ATP is spent during tRNA charging reaction.

  20. Ribosomal RNAs are both structural and catalytic determinants of the ribosome Ribosomal RNAs are not simply structural components but directly responsible for catalytic activity. The peptidyl transferase center and the decoding center are composed almost entirely of RNA. Most ribosomal proteins are on the periphery of the ribosome.

  21. The peptidyl transferase center

  22. The decoding center

  23. The ribosome has three binding sites for tRNA The A site is for the aminoacylated-tRNA. The P site is for the peptidyl-tRNA. The E site is for the exiting tRNA. Each tRNA binding site is at the interface between the large and the small subunits of the ribosome.

  24. Channels through the ribosome allow the mRNA and growing polypeptide to enter and/or exit the ribosome There are two narrow channels in the small subunit, one for entry and the other one for exit of mRNA. only wide enough for unpaired RNA to pass through. There is a kink in the mRNA between the two codons. The incoming aminoacyl tRNA cannot bind to bases immediately adjacent to the vacant A site codon.

  25. A channel in the large subunit provides an exit path for the newly synthesized polypeptide chain. The size of the channel limits the folding of the growing polypeptide chain. The polypeptide can form an alpha helix in the channel.

  26. INITIATION OF TRANSLATION Successful translation initiation needs three events. 1.The ribosome must be recruited to the mRNA. 2. A charged initiator tRNA must be placed into the P site of the ribosome. 3. The ribosome must be precisely positioned over the start codon.

  27. Prokaryotic mRNAs are initially recruited to the small subunit by base-pairing to rRNA The small subunit associates with the mRNA first by base-pairing between the RBS and the 16S rRNA. The small subunit is positioned such that the start codon will be in the P site when the large subunit joins the complex. The large subunit joins its partner only at the very end of the initiation process.

  28. A specialized tRNA charged with a modified methionine binds directly to the prokaryotic small subunit Initiator tRNA: a special tRNA that base-pairs with the start codon (usually AUG or GUG). The initiator tRNA gets charged with N-formyl methionine. The charged initiator tRNA is referred to as fMet-tRNAifMet. Deformylase: removes the formyl group from the amino terminus during or after the synthesis of the polypeptide chain. Aminopeptidase often removes N-terminal methionine as well as one or two additional amino acids.

  29. Three initiation factors direct the assembly of an initiation complex that contains mRNA and the initiator tRNA IF1 prevents tRNAs from binding to the portion of the small subunit that will become part of the A site. IF2 is a GTPase that interacts with three key components of the initiation machinery: the 30S, IF1, and charged initiator tRNA. It facilitates the association of the charged initiator tRNA with the small subunit IF3 binds to the small subunit and blocks it from reassociating with a large subunit. It occupies the E site. With all three IFs bound, the small subunit binds to mRNA and the initiator tRNA in either order. Base-pairing between start codon and the initiator leads to conformational change of the small subunit, resulting in the release of IF3. Binding of the large subunit Hydrolysis of GTP bound to IF2 Release of IF2/GDP and IF1 Formation of 70S initiation complex

  30. Eukaryotic ribosomes are recruited to the mRNA by the 5’ cap In eukaryotes, the small subunit associated with the initiator tRNA is recruited to the 5’ cap and scans along the mRNA until it reaches the first AUG. Four initiation fators (eIF1, eIF3, eIF5, and eIF1A) bind to the small subunit. The initiator tRNA is escorted by eIF2 which forms the ternary complex (eIF2-GTP-charged initiator tRNA). eIF2 positions the Met-tRNAiMet in the future P site, forming the 43S preinitiation complex. The mRNA is separately prepared for recognition by the small subunit. The cap is recognized by eIF4E. eIF4G and eIF4A are then recruited. 4E-BPs compete with eIF4G for the binding to eIF4E. eIF4B activates the RNA helicase activity of eIF4A, which unwinds any secondary structures. Interactions between initiation factors bring the mRNA to the 43S complex to form the 48S preinitiation complex.

  31. The start codon is found by scanning downstream from the 5’ end of the mRNA Correct base-pairing between the start codon and the initiator tRNA changes conformation of the 43S complex and that of eIF5, which stimulates eIF2 to hydrolyze the bound GTP. eIF2-GDP, eIF1, eIF3, eIF5 are then released. eIF5B closely related to IF2 binds the initiator tRNA and stimulates association of 60S subunit. Binding of the large subunit leads to the release of remaining initiation factors by stimulating GTP hydrolysis by eIF5B, forming 80S initiation complex

  32. Translation initiation factors hold eukaryotic mRNAs in circles The poly-A tail contributes to efficient translation. Poly-A binding protein coats the poly-A tail and interacts with eIF4G, circularizing mRNA. The newly released ribosome is positioned to reinitiate translation on the same mRNA.

  33. TRANSLATION ELONGATION The correct addition of amino acids needs three key events to occur. 1. The correct aminoacyl-tRNA is loaded into the A site of the ribosome. 2. A peptide bond is formed between the aminoacyl-tRNA in the A site and the peptide chain that is attached to the peptidyl-tRNA in the P site. 3. The resulting peptidyl-tRNA in the A site and its associated codon must be translocated to the P site. Elongation factors control these events.

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