Protein synthesis in eubacteria In eubacteria such as E. coli, protein synthesis involves three steps ~ initiation, elongation and termination. Initiates involves binding of small ribosomal subunit to mRNA and then is joined by the 50S subunit. It involves the reactions that precede the formation of the peptide bond between the first two amino acids of the protein. After initati: next step is elongation which includes all the reactions from the synthesis of the first peptide bond to addition ¢ the last amino acid. Termination includes the steps that are needed to release the completed polypeptide cha and the dissociation of the ribosome from mRNA. Initiation Initiation of protein synthesis in eubacteria (e.g. E, colf) involves association of small and large subunits of 708 ribosome at the ribosome-binding site (also known as the Shine-Dalgarno sequence) of mRNA. Shine-Delge* sequence is a short nucleotides sequence located 3 to 10 nucleotides upstream of the start codon. It is 2 pure © scanned with OKEN ScannerGentes 213 ev conseneus sequence of aout sx nucleotides 5 -AGGAGGU-Y' upstream ofthe strt con, Lke the consensus ‘eaves n promoters the Shine-Dalgero sequence has a characteristic nucetie comprston and a preie on relative to the stat codon, but ts exact nucleotide sequence varies sight from one mRNA to another. ‘Te sine-Dlgarn sequence ls complementary toa repien a the 3 end ofthe 165 AWA, te ane present the sal svbunt ands thought that base-paing between the twos invlvedin the atachmant ofthe sma subunit tothe mRNA. The extent of complementarity andthe spacing between the Srine-Dalgemo sequence athe start ‘fon ae ® SONG Influence on Now actly a pac ORFs translated: high complementary and Proper _gacing promote Figher level of translation Protein or RNA binding nar the Shine-Oalgarno sequence negatively repultes translation ination, These inhibitors asocate with sequences nea the Shine-Dalgarno Sequence and ‘shyly Inhibit Bese pring between the Shine-Dalgarno sequence and the 165 (RNA, Srine-D850m ——tynaten : euserwetie) Fre 1.182 he shine-Dapr aque mart eno MANA prne-h cosenu eaHens ot Instng amin ald and initiating codon ‘he syntess ofall proteins starts withthe amine acd methionine. In E. coll and in other eubactara, the fst amino acd in any newly syntesized poypeptice s N-formyimetonine, However, formation i not necessary, becnsenonformyated methionine cn function ag aninator amino ac. A formylaton reaction takes place ater ‘methionine Is inked tothe Iibtor RNA, prodcng N4ormyamethonine (atlyzed by enzyne methiony-tRNA fermytvanserse), The source ofthe frry groups -formyetrahydroflae. Eukarotes end archaea also use ‘metvonine ae an ntng amino ai, but isnot frmyloted methionine. Beeuse Nformyimethonine is the fst ‘ina acid tobe Incorporated nt» palypepi cain in eubacteria, one might think tat al eubacteria potens have Yr aroup a thelr amin erin. Tse atte case, awever, Because an enayme deermyase removes the formyl group rom the amino terri during o after the syntass of he polypeptide chan ie econ Fen co, in é ° os by ome emi Figure s.183- Seuss of mon and rms Inewbactera suchas, col, AUG and GUG, and on rar ecasions UUG, serve as ination a tat codons. When ne ofthese tpt a present in the ination poston, f-ormyimathionine appees as the fist amino acd In ‘he chain. the meaning ofthe AUG and GUG codons depends on thelr context. When the AUG code i used for Intinton, ts rad ast formyimethioine: when used within the coding region, I codes methionine. Silay, Srhen GU codon serves axon ination codon, It ls 8d a5 formyimethonne. But when presen wthin the coding reeds vane. th eukaryotes, te inten codon est elaays AUG, @ scanned with OKEN ScannerInitiation factors In addition to MRNA, ribosomes and specific tRNA molecules, initiation requires the participation of several nator factors. Eubacteria use three initiation factors, numbered IF-1, IF-2 and IF-3. IF-1 Binds over the A-site of the small ribosomal subunit and is thought to prevent the initiator tRNA fen binding to the A-site. IF-2 Directs the initiator tRNA to its correct position in the 30S initiation complex. It has a ribosome-depentet GTPase activity. The GTP is hydrolyzed when the SOS subunit joins to generate a complete ribosome IF-3 Binds to free 30S subunits and prevents premature reassociation of the large and small subunits oft ribosome. Its second function is to control the ability of 30S subunits to bind to mRNA. It also ensures‘ fidelity of initiation site selection; select the initiator tRNA for use in initiation. © scanned with OKEN Scannerestes of 13, tel ibn etn te sal nt. Tis, he ere fain the femaon of comet 705 sosome a teresa cing ate fhe mR wh Me NAM™ te Pe gaan eng Pte. Te bosame-mANA complex snow pad to accapt anon RNA complementary 12 sein coton nto the Aste and str pelypeee pane ae ‘Rowe 1185 Eubcesliaton of rotin eyes. Tees nto actos] 2 and) parte Its acess. The 35 stunt Sn ih he PMR, wen ie gaded by ne Glparo sequen. an fhe lp (Ss) nbosomel syne accom rau fe tition sare ang yeas fe -GTF Vale longation engtion i eye process onthe itsome n which one amino aed at atime added tothe nascent peptide chain The peptide seauence is determined bythe order ofthe dons in the mRNA. Elongation rte In Ecole rouphy 15 amino sis er second. The process of angio in exacts aided by te protln actor caled lsongaton factors (EFS), EF-Tu, EF-Ts and EF. Eu (EF-14) Directs the next RNA tots correct poston nthe ebosome 5s (eF-18) __Regonerates EF-Tu ater the hyo of attached GTP molecule (e2) Mediates warslocation. tongetion process, it elongatio factor EF-Tu meats the entry of amlnesey-tRNAS into the Aste io, ey fet binds to GTP (lnary complex formation). The activated EF-Tu.GTP complex Binds to the AANA (emary complex formation). ext, harps ofthe GTP ofthe complex to GOP helps arive te © scanned with OKEN Scannersmerny eee ea ee eevee ono ‘he amino com Pte to Aste genres # dened RNA aching ay amino A) Hes inthe te! ‘rong ere ncaa pequar RNa nese Teen eRMA ee sone vant “estar aaa Desens RMA Rey awe 1.186 the oertyltanteac react. Foraoteme tof th frat ett formed metence {2rd by A presente Pate nt armani he eke apie tod lore nce ee generates a east cng anya te ese Pate, an eset sin tn ste, The ceed eres rosaone ns a eas ‘The lrg 505 bosomal subunit contains the se of aayss—the peti transferase cnter—wich is resent {or mating peptide bonds using protein elongation and forthe hls of peptiaysana dung nc eevee of raten synthe epi warsterese act esis nthe 235 ANA, How des the 236 Rtas pee ‘onc fermation? The exact mechanism remains to be determined, ut some newer tts quater os oer to emerge. Fs, base pong between the 235 WA andthe CCA edo the lain the Kaen pee {the w-amino group ofthe aminoacyH4RNA to attack the carbonyl group of he growing ponpesnos seared ‘eptsy-RlA. This ye of catayte mechanism i called entropic cata, Tat, the exc nons ov br the substrates together ina manner that simuaes cata. ‘Once the peptid! transferase readon hs acute, the Hew peptdARNA nthe A ste asthe ribosome moves exactly tees niceties aang the MANA roca Sing i the 53" sets Ths movements relered to a6 translocation This scp I edad by the elongation cee ete coset transacase) on sven by siting» GT to OP. ug enon arcs hee eens ce gon correct aminoay/-isloaded ithe ste ofthe rare a lated by We hae eos eet ol formed between the arinoac/-4RNA nthe Aste od the gown pete tain has seen est tata in te Pte THs pep nse reacon rest nthe anter tthe pou eaves ene © scanned with OKEN Scannerentice 207 tannin the Pst tothe amino acd moiety ofthe charged tRNA nthe Ait. Finally the resulting petiy-tRNA pri Aste and its asoated codon is Wanslocated tothe Pste so that the ribosome f poses for another cvle nae recognition and peptide bond formation Pe s [yyeamnnnn ge | suas our 1487 ‘Te elongating of roti ayes on 8 reson, The tease le shown repeated over Fa ce ee cptess cs prten can. hn amnaayha lease tnd othe Aste onthe some soos de band sored sie 2, ad te berome moves a dtane of re nee a the chino 3 jectng on dency NA melee destin the bosom so ene AMIDA lease ean bind (© Scanned with OKEN Scanner28 conti Termination rote sytes ens when one fhe hee top codons -UAG (amber), UMA ote) and UGA pay the Aste, At site, stop eadons are read not by spec tRNAS but by spec protens known a reeay 2% Ec ae tvs pt fen as, fairs ET a F2 eso he on ge recagns the sop enn AA gS VAG a RF2 eagles S'UAA an UGA: Rg ater recognizing the stop codons activate the ribosome to hyrolze the Bond between RNA and pan Br nd R2 rece he sap cd and sma poypesise ane OWN 8 6 (oy ‘he ac 5 peptide aon, Ales ot RF3 (eed tothe ong fata EF) site Setanta rom eae rtm mean eg ery on ae Rete factr AF: _Recgnzestn tention con UMA and Uk 3% F2_ Recipies UAR and UCA3: 3 sults dsocston of RF ad 8-2 tom the rosome ater teint. are 1.188 Temi rnin 209 cote, ukaryotes have ony te rele factors; RF, leh ‘ergs a eterna 3M ‘role of eRF-2 is not clear. The release factors terminate a Breventthe subunits rom attaching teeter sh ‘mong prokaryotes, wanton nthe archaea ‘The archaea ribosome snl tothe eat ‘mRNA and the process of transition inka mor closeyresombies the eukaryotes rather than neat ete become (70S). However, the other fetures Ine watt ar similar to eukaryotes tee. An maNA Dearing muertos iknow a a payborme ers vere 4 © scanned with OKEN Scanner