Stepwise Synthesis of Proteins on Ribosomes Notes PDF

rRNA – structural role and a part of a ribosome Stepwise Synthesis of Proteins on Ribosomes  Ribosomes Protein synthesizing machines Binding mRNA and individual aminoacyl- tRNAs to a ribosome increase the efficiency of translation Di...

rRNA – structural role and a part of a ribosome Stepwise Synthesis of Proteins on Ribosomes  Ribosomes Protein synthesizing machines Binding mRNA and individual aminoacyl- tRNAs to a ribosome increase the efficiency of translation Directs elongation at a 3-5 AA/s rate Translation: the process of which a protein is synthesized from the information in mRNA. Elongation is extremely fast.  Prokaryotic cells are used in biotechnology as vessels to replicate the DNA. Both prokaryotic and eukaryotic ribosomes have multiple subunits: RNA and proteins. There is a small subunit at the bottom and a large on the top. Prokaryotic VS Eukaryotic Ribosomes Large unit of rRNA that has a sedimentation rate of 23 Svedberg and 2900 nucleotides. The Eukaryotic ribosome is larger than the prokaryotic one. There are minor subunits that assemble with the larger ones + Large unit of rRNA that small has a sedimentation ones. rate of 28 Svedbergs 60+40=100, but when assembled the sedimentation rate is 80S, because this rate is affected by the size, so when they are assembled the rate is faster than when separate.  Prokaryotic VS Eukaryotic Ribosomes Ribosomal subunits designated in their sedimentation rate in svedbergs Prokaryotic and eukaryotic ribosomes differ in: – The length of RNA molecules – The quantity of proteins associated with it – The size of the subunit The Eukaryotic ribosome is larger than the prokaryotic one.  3D Ribosomal Structure Alpha helices- proteins On top is the large unit , at the bottom is the Net-like shape - rRNA smaller one, in between the mRNA that will get translated passes. There are three locations ( A P E ) where tRNA binds.  The hardest step and the most monitored because any error can cause errors in protein synthesis which either will get Translation Initiation degraded or accumulated until it forms a degenerative disease. There are no correcting factors in the translation as in the transcription that’s why it needs AUG start codon to be carefully monitored. Important for starting in the correct reading frame start with the correct base. Two different methionine tRNAs – tRNAiMET for protein synthesis initiation – tRNAMET to incorporate methionine into a growing chain – Only tRNAiMET can bind in the appropriate location in the p site in the small subunit Usually other tRNAs enter from the A site but the tRNAi AUG codes for one amino acid only as a regulatory step which is the Methionine. is the only one that goes to p There are 21 tRNAs. and binds Each amino acid has a specific tRNA except the methionine which has two. tRNAi MET binds the first MET in the chain for initiation( goes to the AUG in the start).. However there are more METs in the chain ( not just at the start ) so another specific tRNA MET that binds MET within the chain.  Translation Initiation Occurs at the of the mRNA The small and large ribosomal subunits assemble around an mRNA that has an aminoacylated initiator tRNA correctly positioned at the start codon They only assemble if there are mRNAs to translate ( to do function ) , so they assemble when the tRNAi MET (aminoacylated initiator tRNA) binds to the mRNA. Mediation of initiation factors They outnumber the propagation and termination factors because initiation is the most regulated step. Initiation factors stabilize the translation complex, and act as a proof reading switch using GTP hydrolysis Proof reading mechanisms always include hydrolyzing an energy molecule (usually GTP, sometimes ATP) in order to act as a lock of the correct conformation.  Translation Initiation The ones that are separate and not assembled. Initiation factors bind to inactive ribosomal subunits to prevent them from binding where eIF3 binds to 40 s subunit and eIF6 bind to the 60 s large subunit They stay bound to them as long as there is no active mRNA and no tRNAi MET bound to it. Afterwards these Ifs participate in the association of ribosomal subunits once the small subunit is charged with an initator tRNA There are 8 initiation factors that all start with eIF , e= eukaryotic I= initiation F=factor. Prokaryotic initiation factors start with IF only. Translation Initiation Large 60s subunit with eIF6 bound to it 40s small subunit bound to eIF3 After that, the have roles with assembling the initiation unit (down at the end) Initiation end when the ribosome is assembled and tRNAi MET is bound to the AUG codon and located on the P site.  Translation Initiation Preinitiation complex formed from 40 s subunit complexed with multisubunit eIF3 complex which associates with eIF1A and a ternary complex consisting of Met-tRNA and eIF2 bound to GTP GTP bound eIF2 binds Met-tRNA Cells regulate protein synthesis by phosphorylating GDP-bound eIF2 The GTP is going to be hydrolyzed for proof reading Activation and deactivation through phosphorylation ( kinases) and dephosphorylation (phosphatases)  Translation Initiation Back in transcription, RNA got processed in order to change it to mRNA ( 5’ end – methylation , 3’ end-poly A tail) 5’ cap of mRNA is bound by the eIF4 cap binding complex The mRNA eIF4 complex associates with the preinitiation complex through an interaction of the eIF4G subunit of the eIF4 complex with eIF3 on the preinitiation complex Initiation complex is formed So the initiation complex is formed when the preinitiation complex is bound to the mRNA - eIF4 complex  Translation Initiation The ones that are separate and not assembled. eIF1A binds to the small ribosomal subunit in order to fix the tRNAi MET that is bound to eIF2 which is bound to the GTP. This is the preinitiation complex = 40 s subunit + eIF3+ MET tRNAi + eIF2 + GTP + eIF1A The most important thing in the complex is to make sure tRNAi MET has an anticodon that corresponds to AUG. When bound to the ribosome, the mRNA gets scanned in order to the eIF4 binds to the methyl cap on 5’ end of codon AUG and the anticodon to bind. mRNA eIF4 has multiple subunits ( the most There are three sites on the ribosome, important A B G E) A P E , the tRNAi has to bind to the p G subunit binds to the small ribosomal site ( mentioned before) subunit forming an initiation complex Note that the 60 s large subunit is still in the cytoplasm bound to the eIF6.  Translation Initiation The eIF4B subunit of eIF4 performs an architectural role To assemble the eIF4 Positioning the eIF4A RNA helicase subunit so that it can remove short region of RNA secondary structure in bound RNA using Not a proof reading mechanism, ATP is hydrolyzed in order to energy from ATP hydrolysis destabilize the RNA that is in its secondary structure which makes it in the lowest possible energy level so we add energy to destabilize and remove the secondary structure. Initiation complex scans the bound mRNA and stops when the Anticodon tRNAisimet anti codon in the bottom of tRNAi recognizes the start codon Watson-Crick binding This anticodon ( in P site) binds to the first AUG which is the start codon Subunit A is an enzyme ( RNA helicase: they dissociate the secondary structure of the RNA) RNA is a single helix that contains many exposed hydrogen bonds that enable it to form secondary structures and fold upon itself thus won’t be straight. We need the bases of RNA to be exposed (straight) with no secondary structures, that’s why eIF4 contains the helicase to remove them.  Translation Initiation eIF4 at the 5’ end B is structural G binds eIF4 to the 40s subunit A acts as a helicase to remove any secondary structure eIF1A stabilizes the initiation and pre-initiation complexes. - ‫الخطوات مكتوبة قبل‬- They will leave the complex once the anticodon binds to the AUG. GTP gets hydrolyzed which will promote the release of them and to stop scanning. Small subunit with correct binding between the anticodon on the tRNAi and the AUG which makes it ready to bind to the large 60s subunit which is already bound to eIF6. In order for the two subunits to bind, eIF5 hydrolyzes the GTP to dismantle the elF6 from the large subunit to allow it to finally bind to the 40 s small subunit.  Translation Initiation Recognition of the start codon leads to the hydrolysis of the GTP associated with eIF2 to prevent further scanning Initiating AUG is found by being part of the ACCAUGG AUG is a part of the non translated region that is kept in the mRNA without getting spliced out. ( The one discussed in the transcription chapter)  Translation Initiation After GTP bound to eIF2 is hydrolyzed eIF1,2,3,4 dissociate and the large ribosomal subunit unites with the small subunit a process catalyzed with eIF5 and eIF6 by catalyzing a GTP bound to 60 s subunit eIF5 GTP hydrolysis is a proofreading mechanism, making ribosome subunit association an irreversible step  Translation Initiation In order for the two subunits to bind, eIF5 hydrolyzes the GTP to dismantle the elF6 from the large subunit to allow it to finally bind to the 40 s small subunit. This is the complete ribosomal complex with tRNAi MET at the p site and mRNA.  Translation Elongation Elongation factors are required At the completion of translation initiation Met-tRNAiMet is bound to the p site on the assembled 80 s ribosome P site tRNA is linked to the growing polypeptide chain P from polypeptide  Translation Elongation A site – aminoacyl tRNA binds to it. E site- Exit site P site where tRNAi MET goes directly to it, the first MET is bound ( does not enter the A site) and the polypeptide chain will grow as more acids come. Any incoming tRNAs will go through the A site then the P site. The tRNA which contains an aminoacid will go into the A site.. It will induce a conformational change that transfers the amino acid to the MET (1) in the P , ‫طريقة االضافة مشروحة بعدين‬ Then the tRNA ( which is empty now) will go to the E site where it exits the ribosomal complex.  Translation Elongation The second aminoacyl-tRNA is brought into the ribosome as a ternary complex in association with EF1α. GTP and becomes bound to the A site EIt here stands for elongation has multiple subunits, the alpha part is bound to GTP A site given the name because Aminoacyl- tRNA binds Translation Elongation A site is open. tRNA is going to enter the A site, then its going to see if the anticodon in the bottom of it matches the codon of mRNA that is exposed in the A site. If it matches, the elongation process continues , if not it will exit and another tRNA comes in and checks if there is a complementarity between the codon and the anticodon and so on until the appropriate tRNA locks in and the binding happens though Watson-Crick binding.  Translation Elongation EF1α.GTP bound to various aminoacyl-tRNAs diffuse into the A site but the next step in translation proceeds only when the tRNA anticodon base-pairs with the second codon in the coding region When the codon and anticodon base-pair the GTP bound to the EF1α.GTP is hydrolyzed promoting a change in the ribosome leading to tight binding of AA-tRNA in the A site and release EF1α.GDP GDP because GTP is hydrolyzed. Translation Elongation Once tRNA anticodon base pairs with the codon in the mRNA, GTP gets hydrolyzed which provides energy to change the conformation of the ribosome, Ef1alpha+GTP+P leave the complex and tRNA on the A site is closely bound to the ribosome.  Translation Elongation The ribosomal conformational change also positions the aminoacylated 3’ end of the tRNA in the A site in close proximity to the 3’ end of the Met-tRNAiMet in the P site GTP hydrolysis does not occur if the anticodon of the incoming aminoacylt-tRNA cannot base-pair with the codon at the A site And no conformational change happens.  Translation Elongation MOST proofreading GTP hydrolysis by EF1α is another mechanisms are associated with proofreading step that allows protein energy consumption. synthesis to proceed only when the correct aminoacylated tRNA is bound to the A site The α amino group of the second amino acid reacts with the “activated” (ester-linked) methionine on the initiator tRNA, forming a peptide bond tRNA activating is done by aminoacyl tRNA synthetase , we consume energy to create a high energy ester link between the amino acid and its corresponding tRNA which will be the energy source for creating the new bond between two consecutive amino acids. Hence, there is no direct ATP consumption when creating the peptide bond between the amino acids in the ribosome because ATP was already consumed in making the ester link. Translation Elongation The proximity between the two amino acids 1 and 2 will cause 1 (MET) to break the ester link to form a lower energy peptide bond with the next amino acid. 1 moves to 2 on A site.  Translation Elongation The first amino acid is going to be at the end.  Translation Elongation Peptidyl transferase reaction is catalyzed by the large rRNA which precisely orients the interacting atoms permitting the reaction to proceed After the peptide bond formation the ribosome is translocated along the mRNA a distance equal to one codon Translocation is monitored by the hydrolysis of the GTP in eukaryotic EF2.GTP One codon = 3 bases This is a very sensitive move , any error may lead to frame shift Any translocation step needs the hydrolysis of GTP  Translation Elongation Once translocation occurs correctly the bound GTP is hydrolyzed which prevents the ribosome from moving along the mRNA in the wrong direction or from translocating an incorrect number of nucleotides It will move from 5’ to 3’ end Resulting from the conformational changes in the ribosome that accompany proper translocation and the resulting GTP hydrolysis by EF2 tRNAiMet is moved to the E site Hydrolysis causes translocation and the ribosome to change into its original conformation before the hydrolysis of EF1 During translocation, tRNA on the A site, including pp chain that has moved to it, moves to the p site. MET tRNAi that has no longer any amino acid bound to it moves to the E site. The conformation of the ribosome changes back the way it was before the hydrolysis of the GTP that was bound to EF1. This opens the A site in the ribosome that allows a new tRNA to enter the ribosome and do another base pairing and etc…  Translation Elongation Once the uncharged ( no amino acid) MET tRNAi reaches it, it will be released into the cytosol then E site is the Exit site recharged with the amino acyl synthetase Simultaneously the second tRNA bound to polypeptide chain is moved to the P site Translocation returns the to a state in which the ribosome conformation where the A site is open and able to accept another aminoacylated tRNA complexed with EF1α.GTP beginning another cycle of chain elongation Translation Elongation Process mentioned before. Translation Elongation Ribosome Structure Scanning electron microscope Ribosome Structure 3D rendering  Translation Termination Termination is assisted by release factors when a stop codon is reached Eukaryotic eRF1 has a shape similar to a tRNA and binds to the stop codon in the ribosomal A site eRF3 is a GTP binding protein which acts with eRF1 to promote cleavage of the peptidyl- tRNA releasing the complete protein chain For termination we have release factors eRF – eukaryotic release factor. We have 3 different stop codons , once one of them is reached, eRF1 is bound to it in the A site. eRF3 releases the pp chain from the p site.  Translation Termination UAA is one of the stop codons eRF1 binds to the stop codon GTP on eRF3 gets hydrolyzed which will produce energy that dissociates the growing pp chain from the tRNA in the p site. eRF1+ eRF3+GDP get released The ribosomal subunits disassemble unless there is another mRNA molecule coming. Translation happens to more than one mRNA molecule or to the same mRNA molecule more than once.  Translation Termination Bacteria have two release factors (RF1, RF2) that are functionally analogous to eRF1 and eRF3 eRF3 GTPase monitors the correct recognition of a stop codon by eRF1 Hence its bound to a GTP so it’s a proof reading mechanism Peptidyl-tRNA bond of the tRNA in the P site is not cleaved until one of the three stop codons is recognized and once the GTP on RF3 is hydrolyzed.  Translation Termination Additional release factors promote the dissociation of the ribosome And also promotes the binding of initiation factors eIF3 and eIF6 to the ribosomal subunits to make them stay disassembled.  GTPases eIF2.GTP is hydrolyzed to prevent further scanning of mRNA Once the first AUG start codon is found eEF2.GTP leads to correct translocation of the ribosome along the mRNA eRF3.GTP assures correct termination By reaching a correct stop codon.  Enhancing Translation Simulataneous translation of a single mRNA molecule by multiple ribosomes (polyribosomes/ polysomes) Poly(A) binding protein I (PABP1)interacts with mRNA poly A tail and the 4G subunit of eIF4 which binds to the 5’ end of the mRNA Multiple ribosomes found in the same location one SO a single mRNA molecule get translated into more than one copy of the protein. To prevent that, PABP1 binds to the poly A tail at the 3’ end of mRNA molecule and to the 5’ end making a circle.  Enhancing Translation SEM of a polyribosome Poly A tail at 3’ end 5’ end is methylated and bound to eIF4 E and G. Subunit G which is in the end of the mRNA molecule binds to the PABP1 making a circular structure of mRNA where multiple ribosomes bind , making copies of the protein.  Enhancing Translation Rapid recycling of ribosomal subunits after they disengage from the 3’ end of an mRNA They don’t always bind to eIF3 and eIF6 instead if there are other initiation factors another pre-initiation complex forms and the ribosome starts translating either the same mRNA molecule or another one.