BS2004 Lecture 2: Translation
BS2004 Lecture 2: Translation BS2004
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This 3 page Class Notes was uploaded by Loges Gobi on Monday October 5, 2015. The Class Notes belongs to BS2004 at Nanyang Technological University taught by in Summer 2015. Since its upload, it has received 37 views. For similar materials see Molecular and Cell Biology in Biological Sciences at Nanyang Technological University.
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Date Created: 10/05/15
LECTURE 2 TRANSLATION RNA TO PROTEIN Ribosome has 3 binding sites for tRNA 1 E site exit 2 P site peptidyltRNA binding site 3 A site aminoacyltRNA binding site Initiation factors IF1 assist binding of IF3 to 30s subunit binding also occludes the A site domain of small ribosomal subunit IF2 IFZGTP binds the initiator VlettRNA IF3 binds to 30s subunit freeing it from its complex with 50s subunit enabling 30s to bind mRNA mRNA binding aids correct positioning of complex such that fMettRNA interacts via base pairing with AUG initiation codon must be released from 30s for 50s to join Elongation factors EFTu EFlA binds and delivers an aminoaccyltRNA to A site EFTs EFlB functions as a GEF to reactivate EFTu EFG EFZ facilitates translocation of ribosome relative to mRNA Release factor RF1 RF2 RF3 Ribosome recycling factor RRF GTP binding proteins has different conformation depending on whether it has bound to GTP or GDP bound GTP stabilizes ACTIVE conformation reactivation occurs by release of bound GDP in exchange for GTP via guanine nucleotide exchange factor GEF 1 Initiation IF3 binds to 30s subunit IF1 assists binding of IF3 and also occludes A site IF2GTP binds the tRNAfMet and docks with the 30s 30s preinitiation complex together with tRNAfMet binds the mRNA guided by the ShineDalgarno sequence gt AUG sequence located from which protein initiation begins Addition of large 50s subunit is accompanied by release of protein factors and hydrolysis of IF2GTP to yield complete 70s initiation complex charged initiator tRNA fMettRNAiMet is positioned in P site 50s serves as GAP GTPase activating protein for IF2GTP ribosome now ready for entry of next aminoacyltRNA at A site which will ead to formation of first peptide bond Elongation 2 Elongation Codon recognition gt anticodon of an incoming aminoacyltRNA base pairs with complementary mRNA codon in A site hydrolysis of GTP increases the accuracy and efficiency Peptide bond formation gt rRNA molecule of large subunit catalyzes formation of peptide bond between new amino acid in A site and carboxyl end of growing polypeptide in P site gt attaches the polypeptide to tRNA in A site Translocation gt ribosome translocates the tRNA in A site to P site empty tRNA in P site is moved to E site where it is released mRNA moves along with its bound tRNAs bringing the next codon to be translated into A site EFTuGTP binds and delivers aminoacyltRNA to A site loaded tRNA must have the correct anticodon to base pair with the mRNA codon that is positioned in A site gt tRNA binding causes conformational change in small subunit gt universally conserved bases of 16s rRNA interacts closely with minor groove of first 2 base pairs of codonanticodon complex gt occupancy by incorrect tRNA interaction is not stable and aminoacyltRNA is released proofreading EFTuGTP is hydrolysed to EFTuGDP when aminoacyltRNA is delivered to A site a domain of the ribosome functions as GAP for EFTu gt large conformational change in EFTuGDP promotes dissociation of EFTu gt release of EFTu leads to repositioning of aminoacyltRNA to promote peptide bond formation EFTs function as GEF to reactivate EFTu release GDP from EFTu Translocation of ribosome relative to mRNA involves EFGGTP binds to ribosome in vicinity of A site gt binding may push the tRNA with attached nascent polypeptide from A site to P site Frees up A site ribosome functions as GAP for EFG Transpeptidation involves acid base catalysis by a universally conserved adenosine of the 23s rRNA ribozyme as it has no protein component gt amino N of amino acid that is linked to 2 or 3 OH of the terminal adenosine of tRNA in A site reacts with the carbonyl C of amino acid linked to tRNA in P site gt H donated to the hydroxyl of tRNA in P site as the ester linkage is cleaved gt nascent polypeptide is now linked to tRNA in A site translocation has partly occurred as peptide bond formation is associated with rotation of single stranded 3 end of A site tRNA towards the P site and it also positions the polypeptide to feed into entrance of protein exit tunnel 3 Termination When ribosome reaches a stop codon on mRNA A site of ribosome accepts release factor instead of tRNA gt release factor hydrolyzes bond between tRNA in P site and last amino acid of polypeptide chain gt 2 ribosomal subunits and other components of the assembly dissociate RF1 and RFZ recognize and bind to stop codons RF3GTP facilitates binding of RFl and RFZ to ribosome ribosomal peptidyl transferase catalyzes transfer of peptidyl group to water hydrolysis gt RF3 GTP to RF3GDP causes conformational change that results in dissociation of release factors Ribosomal recycling factor required with EFGGTP and IF3 for release of uncharged tRNA from P site and dissociation of ribosome from mRNA with separation of 2 ribosomal subunits Doublesieving mechanism 1 Size exclusion enzyme first binds similar or correct substrate and reject others correct amino acid has highest affinity for active site pocket of its synthethase 2 incorrect adenylated amino acid is hydrolysed at the 2nd editing tRNA docking onto synthethases forces the wrong adenylated amino acid into a second pocket editing pocket of protein gt hydrolysis of adenylated amino acid Kinetic proofreading model initial selection phase codon recognition in which the tRNA anticodon associates reversibly with the codon in the 30s A site gt recognition of cognate codon and anticodon leads to stabilization of ternary complex on ribosome and facilitates GTPase activation of EFTu gt EFTuGDP leads to conformational change in ribosome gt either aminoacyl end of tRNA moves into A site of 50s leading to peptide bond formation or incorrect aa tRNA dissociates from ribosome Wobble hypothesis pairing between codon and anticodon at first 2 codon positions always follow usual rules but exceptional quotwobblesquot occur at 3rd position gt conformation of tRNA anticodon loop permits exibility at first base ofan codons Inducedfit model conformational change to ribosome gt domain closure in the 30s subunit induced by cognate tRNA binding accommodation vs rejection New proofreading mechanism RFZ catalysed release factor for P site mismatched complex gt after formation of peptide bond P site mismatches compromise tRNA selection fidelity