BMB 462 Lecture 35 Outline of Last Lecture I. Ensuring Specificity of tRNA ChargingII. Proofreading by aminoacyl synthetasesIII. Ribosome structure and assemblyIV. The ribosome as a ribozymeV. Initiation of translationVI. Initiation complex in EukaryotesVII. The 1st step of elongationOutline of Current Lecture I. Continuing the 3 steps of elongationII. Termination of translationIII. Translation factors used by the cellIV. Coupling transcription and translationV. Chaperones and posttranslational modificationsVI. Inhibitors of translationVII. Translocating proteins across a membraneCurrent LectureConcepts 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 proofreadthe 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 elongationa. 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 E-site (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 A-site 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 translationa. 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 theA-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 cella. IF-2, EF-Tu, and EF-G are all GTPasesi. 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 translationa. 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 nucleusthat 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 modificationsa. 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 α-helices and the β-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. Deformylation1. 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. Acetylation1. Addition of an acetyl group to an
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