BMB 462 Lecture 35 Outline of Last Lecture I Ensuring Specificity of tRNA Charging II Proofreading by aminoacyl synthetases III Ribosome structure and assembly IV The ribosome as a ribozyme V Initiation of translation VI Initiation complex in Eukaryotes VII The 1st step of elongation Outline of Current Lecture I II III IV V VI VII Continuing the 3 steps of elongation Termination of translation Translation factors used by the cell Coupling transcription and translation Chaperones and posttranslational modifications Inhibitors of translation Translocating proteins across a membrane Current Lecture Concepts to remembers from previous courses lectures Step one of Elongation involves EF Tu bringing in a new aminoacyl tRNA to the A site of the ribosome While the EF Tu a GTPase is hydrolyzing GTP the ribosome can proofread the tRNA and make sure the tRNA with the right anticodon has been bound If it is incorrect to the current codon the tRNA is released This is a type of kinetic proofreading I Continuing the 3 steps of elongation a Step one of elongation ends with the original aminoacyl tRNA in the P site and the new aminoacyl tRNA bound to the A site but no peptide bond has been formed b Step two forms that peptide bond These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute i The 23S rRNA catalyzes the reaction for forming a peptide bond between the two aminoacyl groups ii When the tRNA is charged with an amino acid it has a high energy ester linkage This linkage can now undergo a nucleophilic attack by the amino group of the second amino acid and a peptide bond is formed 1 The first amino acid is then transferred to the second tRNA creating dipeptidyl tRNA in the A site of the ribosome 2 The first tRNA in the P site is de acylated c Next the de acylated tRNA is removed from the P site and the next tRNA is transferred from A to P site translocation is the third step of elongation i EF G aka the translocase is the elongation factor that aids in moving the tRNA from the A site to the P site EF G does this by binding to the A site kicking out the de acylated tRNA from the P site and moving the next tRNA from the A site to the P site 1 The de acylated tRNA temporarily moves from the P site to the Esite Exit site and then is released 2 Now in the ribosome you have a tRNA with two amino acids bound in the P site and the A site is free to accept the next aminoacyl tRNA ii EF G is another GTPase it slowly hydrolyzes GTP while bound to the Asite and then is released Removal and translocation of the 2 tRNAs occurs during this GTP hydrolysis 1 EF G can bind to the A site because it mimics the appearance of an EF Tu tRNA complex So after binding to the A site since it doesn t have an amino acid it s not a tRNA it essentially just translocates over the actual tRNAs and then departs d Initiation occurs just once for each polypeptide but the steps of elongation are performed many times to make a longer and longer peptide e The process of translation is very costly it requires breaking 4 high energy bonds to form 1 peptide bond i 2 high energy bonds are broken to charge the tRNA Then 2 high energy bonds in GTP are hydrolyzed during the 3 steps of elongation ii This does not include the bond that is broken during initiation because that only occurs once in the translation process iii The high energy cost is also used towards ensuring fidelity because the time needed for hydrolysis of GTP is used for proofreading II Termination of translation a When there is a stop codon in the mRNA the regular pattern of elongation is interrupted by a release factor which recognizes the stop codon and binds to the A site in the ribosome i The release factor binding to the A site changes the peptidyltransferase activity of the ribosome The peptidyltransferase is typically what makes the amino peptide bond but when the release factor binds there is no new amino acid to be added Instead the last amino acid is hydrolyzed from the tRNA and the peptide chain is released b In the very last step of termination and translation the subunits of the ribosome dissociate so they can be recycled and participate in another round of translation III Translation factors used by the cell a IF 2 EF Tu and EF G are all GTPases i Binding and hydrolyzing GTP changes the conformation of the enzyme in the GTP bound form and GDP bound form the enzymes have a different conformation ii The GTPases tend to bind to their targets better in the GTP bound form than in the GDP bound form so in a reaction the GDP bound form is released IV Coupling transcription and translation a In bacteria transcription and translation are coupled This is because bacteria do not have a nucleus to separate the mRNA produced in transcription from the ribosomes i In eukaryotes the two processes are separated because there is a nucleus that prevents the mRNA from being immediately accessible to the ribosome Transcription is compartmentalized in the nucleus while translation remains separated in the cytosol of the cell b In eukaryotes and bacteria there are often more than one ribosome translating the same mRNA simultaneously this complex is called a polysome i It is possible to isolate mRNAs that are associated with a polysome thus allowing insight into the types of mRNAs that are currently being translated in the cell V Chaperones and posttranslational modifications a Now that a protein has been translated it must be properly folded into its secondary and tertiary structures i The secondary structure is composed of all the helices and the sheets with primary structure referring solely to the amino acids incorporated into the chain ii Tertiary structure refers to the 3D folding of the protein 1 This folding is usually only accomplished in the presence of chaperone proteins There are chaperones in the cytosol that aid the polypeptides to fold in their correct structure 2 Some proteins especially smaller proteins can typically fold on their own iii Quaternary structure in proteins refers to different subunits that associate b Posttranslational modifications i Deformylation 1 In bacteria all of the N termini of proteins are formed with a formylated methionine which has to be removed before the polypeptide is complete ii Acetylation 1 Addition of an acetyl group to an amino group in the protein Often it is added to the N terminus approximately half of all eukaryotic proteins have
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