Protein SythesisTranslation課件

1、protein synthesis-translation concept of translationprotein synthesis requires: mrna、rrna、trna substrates: 20 amino acids enzymes and protein factors: initiation factor, elongation factor, releasing factor atp、gtp、mg2+ prokaryotic mrna is polycistron. eukaryotic mrna is monocistron.the genetic code

2、the genetic code is a triplet code 64, more than required, but not overly excessive three codons for stop signal: uaa, uag, uga. 61 codons for 20 amino acids. one codon for start signal: aug. it also codes for methionine.nonoverlapping and without punctuation open reading frameframeshiftdegenerate,

3、but unambiguous synonymous codons ? more fault-tolerant for point mutations. wobblebase pairs of inosine fewer than 61 trnas are required enhances the efficiency of protein synthesis the code is almost universal all organisms use the same genetic code.cytoplasm aua: ile aug: met, initiation uaa, uag

4、, uga: terminationmitochondria aua: met, initiation uga: trp aga, agg: termination however, there is intriguing exception of a few minor variations. trna and aa activationtrnaactivation of amino acidactivated amino acid ala-trnaala ser-trnaser met-trnametsummary of aa activation active form aminoacy

5、ltrna activation site - carboxyl group linkage ester bond activation energy 2 high-energy bondsprotein synthesis fidelity aminoacyl-trna synthetase has the proofreading ability. the overall error rate for translation in e. coli is only 1 per 50,000 codons.prokaryotic met-trnamet prokaryotic met-trna

6、met can be formylated to fmet-trnaimet. met-trnamet + n10-formyl tetrahydrofolate formyl transferase fmet-trnaimet + tetrahydrofolate initiation codons for prokaryotes: fmet-trnaimet initiation met-trnaemet elongation. for eukaryotes: met-trnaimet initiation. met-trnaemet elongation. recognized by e

7、nzymes or factors in different phases.ribosome of prokaryotesthree sites on ribosomeslocationfunctionaminoacyl site(a site)composed bylarge and smallsubunitaccepting anaminoacyl-trnapeptidyl site(p site)composed bylarge and smallsubunitforming thepeptidyl bondsexit site(e site)only on largesubunitre

8、leasing thedeacylated trnaa site, p site and e site the aminoacyl (a) site, the peptidyl (p) site, and the exit (e) site translation in prokaryotes initiation three steps in prokaryotic systems positioning mrna on the 30s subunit registering fmet-trnaimet on the p site associating the 50s subunit th

9、ree initiation factors: if-1, if-2 and if-3. in prokaryotes, the two types of trna specific for methionine are designated trnafmet and trnamet. the trnafmet is used for the aug initiation codon and is called the initiator trna. shine-dalgarno sequence an initiation signal of purine-rich the 3end of

10、16s rrna has consensus sequence uccu which is complementary to agga in s-d sequence (also called ribosomal binding site).initiation 1 the if-1 and if-3 bind to the 30s subunit. t h e m r n a t h e n binds to this subunit. the 16s rrna is aligned with mrna, allowing aug on the p site.initiation 2 the

11、 complex of the gtp-bound if-2 and the fmet-trna enters the p site.initiation 3 the 50s subunit combines with this complex. gtp is hydrolyzed to gdp and pi. all three ifs depart from this complex. note that: unlike all other aminoacyl-trna molecules which bind the a site, the binding of fmet-trnafme

12、t occurs directly into the p site. elongation three steps in each cycle: positioning an aminoacyl-trna in the a site forming a peptide bond translocating the ribosome to the next codon elongation factors (ef) are required.ribosomal cycle: the peptide chain is extended by one amino acid as the riboso

13、me moves one codon downstream on mrna. the elongation cycles continues until the stop codon enters into the a site.step 1: entrance an aa-trna occupies the empty a site. registration of the aa-trna is an energy consuming process. the entrance of aa-trna needs to activate ef-t. the energy is provided

14、 through a hydrolysis of gtp.ef activation cycle ef-t is composed of tu and ts subunits. tu has two active sites, one for gtp and another for aa-trna. tu is gtpase. tu cannot bind to the initiating aa-trna.peptide bond formation 1peptide bond formation 2 peptidyl transferase is requiredtranslocation

15、 1 ef-g is a translocase. gtp bound ef-g provides the energytranslocation 2 the dipeptidyl-trna is now entirely in the p site, and the a site is open for the next incoming aa-trna.termination prokaryotes have 3 release factors: rf-1, rf-2 and rf-3. rf-1 and rf-2: recognizing the termination codons r

16、f-3: gtp hydrolysis and coordinating rf-1/rf-2 and rpstermination 1 uaa and uag: rf-1; uaa and uga: rf-2.termination 2 the peptidyl transferase is converted to an esterase. the ester bond of polypeptidyl-trna at the p site is hydrolyzed, and the nascent peptide is released .termination 3 the uncharg

17、ed trna, mrna, and rfs dissociate from the ribosome. the whole process of protein synthesis finishes.energy consumption initiation： one gtp (if-2-gtp) aa activation：two p bonds elongation： two gtp (tu-gtp, ef-g-gtp) termination： one gtp (rf-3)polysome proteins are synthesized on a single strand mrna

18、 simultaneously, allowing highly efficient use of mrna.animationinterference of translation the protein synthesis is highly regulated. this process can also be the primary target for many toxins, antibiotics and interferons. these interferants interact specifically with proteins and rnas to interrup

19、t the protein synthesis. the interference leads to the death of bacteria for clinical treatment.antibioticsnametargetfunctiontetracycline30sblock the a site to preventbinding of aa-trna with 30sstreptomycin30srepress the translocasechloromycetin 50sblock the peptidyl transferase,and inhibit the init

20、iationerythromycin50sinhibit the translocasecycloheximide 60srepress the translocase,inhibit the elongationpuromycinrelease the prematuredpeptidechemical structurespuromycin aminoacyl-trna analog, binds at a site and acts as peptidyl acceptor, aborting peptide elongation. it works for both prokaryot

21、es and eukaryotes.toxins other toxins, such as plant protein ricin, is among the most toxic substance known, which acts on 60s subunits. they apparently catalyze some modification of a ribosomal protein but the chemical nature of the alteration is unknown.diphtheria toxininterferon the initiation is

22、 a rate-limiting step. biologically active biomolecules affect the protein synthesis. interferons are cytokines produced during immune response to antigens.interferonprotein modification andprotein targetingprotein foldingmodification of primary structure removal of the the first n-terminal methionine residue covalent modification of some amino acids (phos