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    346 csusrenw | Regulation of Bactarial Celular Processes Forespore @-e. ‘Septum 6 kina kn soe Shr orks Shee sxfon Sean ® Eatly sporlton ‘Active of ——t one vanserption Late poration ‘gene transcristion| Aetiveo8 ® Figure 14.28 Regulation ofSporuation in, subtilis: The ntation of sporulation s governed in party the ates of two spatial separated sigma factor. os cated inthe forespore, whe i confined othe mathe cel These sigma factrs cect the natin of tansciton of genes whose procucts are neeced for early evens sporllion Laer, a nda are localize one developing endosoore and mether cel especvey They coll he ‘expression of genes whose products are nvoledin ater steps of sparlton. fo} The activation ofa is accomplished troughs phosohoreay system hats ‘tggered by the acwaton of the sensor kinase protein KinA When Kin senses starvator, i autophospharylats specifi hstine residue The phosphoryl ‘grup s tren passin relay fasion rom Sp00F to Spo08 ac nly to Spo0R MICRO INQUIRY Compartmentston—the spt loolztion of pres aso means ofregulaton—i uso considered inthe corer of eukaryotic es. Hows the proces fspealton an example of comparimentaton? subtilis finds its nsients ae depleted, KinA autophosphoryates 2 specific histidine residue. The phosphoryl group is then tane~ ferred to an aspartic acid residue on SpoO, the second member of the phosphorelay. SpoOF donates the phosphoryl group to a histidine on Spo03. Spo0B in tu relays the phosphoryl group to Spo0A. Phosphorylated Spo0A positively controls genet needed for sporulation and negatively contols genes that ate not needed. In response to SpoOA, the expression of over 500 genes is altered. This has earned SpoOA the name “master regulator” ‘Among the genes whose expression is stimulated by SpOOA is sigF, the gene encoding 0”, and spolIGB, the gene encoding an inactive form of o (pro-o4) ‘When sporulation starts, the chromosome has replicate, with one copy remaining inthe mother cell and another to be partitioned in the forespore. Shorly after the formation of the spore septum, 0” is found in the forespore, and pro-o" is localized to the mother cell. Pro-a is cleaved by a protease to form active o®. The two sigma factors, o” and o®, bind to the promoters of genes needed in the forespore and mother cell, respectively. There they direct the expression of genes encoding products needed for the early steps of endospore formation. These genes are primarily responsible forthe en ‘guifiment process. Another gene regulated by o” is one that encodes 0°, which will replace o” in the developing endospore. Likewise, directs the wanscrption of a mothercell-specfic sigma factor, o¥, o¥ ensures that genes encoding late-stage sporulation products are transcribed, These include genes for synthesis ofthe cortex and coat layers of the endospore. Over all, temporal regulation is achieved because a and 0” dizect \wanscription of genes that are needed early in the sporulation process, whereas o° and o® are needed forthe transcription of zenes whose products function later. In addition, spatial con- trol of gene expression is accomplished because o® and 0 are located in the forespore and developing endospore, whereas oF and o* are found only in the mother eel. Retrieve, Infer, Apply 1. What woul be the phenotype of fice mar hat cous nt rete that twas constant producing atnducer thigh tet? 2. Why mint acters use quorum sensing to regu genes needed forwlence? How might season be related the ratonale Behind sng quota sersng to estbish a sybiteretonsip? 2. Whyis the stingent response ypcalyacve ring nitrogen starvation ut ot inrespone catbon sources? 4. Brey describe how a phosphoely system and sigma factors are sso canto spruton Bsus. ie one example of postranlaonal medication sa means oreglat is process. pts 1 Bacteria Use Many Regulatory Options 1 Regulation of geae expression can be controlled st many levels including anscription initiation, ranscription ‘Archaeal DNA, polymerase D (Pol D) Repiicative DNA polymerase in some ‘archaea; Unigue to ‘rehaea x Eukaryotic DNA, DNA repair polymerase 8 y ‘Bacterial NA DNA repair polymerase IV (Ding) Reverse __—Retrouralreverse —-_—RNAdependent DNA ransciptase transcriptases polymerase en) Telomerase RT Telomeres and Telomerases: Protecting the Ends of Linear DNAMolecules ‘The fact tha the DNA in eukaryotic chromosomes is linear poses several problems, Without protection, the ends are susceptible to degradation by enzymes called DNases. They ate also able to fuse with the ends of other DNA molecules, thus generating aberrant chromosomes. Finally, linear chromosomes present a problem during replication because of DNA polymerases aced for a primer that provides a free 3-OH. When the primer forthe Okazaki fragmenta the end of the daughter strand ie removed, the daughter molecule is shorter than the parent molecule. Over numerous rounds of DNA replication and cell division this leads to. progressively shortened chromosome. Ukimatey the ehro- rotome loses critical genetic information, which could be lethal tothe cell. This is called the “end replication problem,” and a cll rust solve it ft isto survive Bukaryotc cells have solved the diffcuies related to hav- ing linear chromosomes by forming complex structures called telomeres (figure 15.3) atthe ends oftheir chromosomes and by using an enzyme called telomerase (igure 15.4) Telomeres are protein-DNA complexes tha protec the linear DNA within them from degradation and end fasion. The protein component of a telomere varies from species to species, a does the length of DNA present. Telomeri DNA contains many capes ofa partic- ular sequence of nucleotides, placed one after the other adem 15.2 DNA Replication: Similar Overall, but with Diferent Rephsome Proteins 352 (2) cllates Figure 15.3 Telomere Suuctue. (e) Fission yeasts Telomeres consis ofthe end of te NA molecule complexed th proteins. The eaton, number, an nature ofthe pots fer tom eegansm 0 ogansm, or cach elomare iste identical proteins are show having the same shape. iterent shapes inca tat the protein isnot the same. Nol at some proteins bnd te srg sanded al, whereas cers bind upstream where theDNA's double sanded Telomere ofthe ciate pots “Sonia and Onicha spp) Telenares of buen yeass such 5 Socchronyces corse. Telemere of ison yeast such as Seizosoccharamjces pombe, “Telomere “Teloreres have repeated sequences that ae complementary 0 inteal RNA erste of the telomerase enzyme Therefore, the internal NA tomplate base-pars Ropes unt whetey toate —— Shut staned porto Teaser Tsasa oie loners ERACECCARG Intl RNA template “The reverse transcriptase actly of telomerase Uses the internal RNA Telomerase 2 2 Grnucleotde repost, TSGCCTTSGSS! satenaite or igteng te sge TEAGCECAAG Sa oe lome rucleties fo the right and ‘Telomerase moves & sins to make another repeat. ‘The complementary ‘stand ie made by primase, DDN polymerase, and igase When the single stand he telomere long ‘enough, primase Erresserraaa Snape ser ecaaceeca er wow x poner sytend Space Figure 15.4 Telomerase Repicates the Teomeric DNA of Eukaryot Chromosomes Telomerase contains an NA mlecul thet can base-ai wth a sal partion of the 3" overhang {the Gai The RNA serves sa template or DNA syrthesis atazod bythe reverse Wansripise ‘eivty ofthe enayme, The 2-OH ofthe tele ONR serves a ne arimer andi lengthened. The process shown s repeated may ines urithe 3 overhangs long enough o serv asthe ‘emplate for synthesis ofthe commentary telomere DNA stranby te repsome repeats). Importantly, the DNA is single-stranded atthe very end and is called the G-tail because it i rich in guanosine bases; telomerase uses the ‘Geta to maintain chromosome length ‘Telomerase has two important components: an intemal RNA template and an enzyme called telomerase reverse transcriptase (RT), The internal RNA template is complementary toa portion of the ‘Getail and base-paits with it Gigute 15,4). The ternal RNA template provides the template for DNA synthesis, which is catalyzed by telomerase RT Ge, the 3-O ofthe Gail serves asthe primer for DNA synthesis. After being lengthened suff- ciently, there is room for synthesis of an RNA primer, andthe single stand of telomere DNA can serve as the template for synthesis of the eomple- ‘mentary strand. Thus the length of the chromo- some is maintained, ‘Telomerase RT deserves additional com- ‘ment. As described in the preceding paragraph. telomerase RT synthesizes DNA using an RNA template, Enzymes with this capability are de- fined as RNA-dependent DNA polymerases. RNA- dependent DNA polymerases are not unique to telomerases; certain viruses use RNA-dependent DNA polymerases to complete their life cycles (eg, human immunodeficiency viruses and hepa- itis B viruses). These interesting viruses are described more in chapters 27 and 38, Archaea Use Homologues of Eukaryotic Replisome Proteins ‘The mixture of bacterial and archaeal features is ‘bvioss when archaeal genomes are examined Archaeal chromosomes ae similar in size to those of bacteria, Inaditon, all known archaeal chromo- somes age ccc, like mos bacterial chromosomes However, ome archaea hive histones associated ‘wth ter chromosomes ust ike eukaryotes. DNA replication in archaea also illustrates their “hybrid” nature The circular ebromosomes of archaea are replicated by replisomal proteins that 354 csuprens | Eukaryotic anc Archaeal Genome Replication and Expression ate similar to those of eukaryotes (figure 15.5; table 15.1). Most archaeal cells have a single origin of replication, like bacterial chromosomes; however, a few have more than one. The reason for this is unknown, as archaeal chromosomes ae not especially larg. However, having more than one origin may be related tothe speed at which the replisome travels around the DNA, Those with faster speeds have single origin, whereas those with slower speeds have smioge than one oxigin, Thus having multiple origins may serve the same purpose as in eukaryotes: decreasing the time needed to rep- licale the chromosome, Analysis of archaeal primases and replicative DNA poly- rerases has led to some intriguing discoveries. Nearly all a= chaeal primases are heterodinsevc; that is, they ate composed of | ‘wo differeat protein subunits, PriS and Pri, just like their cukaryotic counterparts. One (Pri) isthe catalytic subunit and the other (Pril} is thought to function as a regulatory subunit. ‘Archaeal primases vary dramatically in terms of their activity. Some are thought to make RNA-DNA hybrid primers as in eu- karyotes, Others appear to make RNA primers, Furthermore, some archaeal primases have functions other than priming DNA synthesis, including filling gaps as part of DNA repair processes. (ther archaea have monomeric primases consisting of both PriS and Pril. sequences fused into a single polypeptide. Archaeal DNA polymerases fll into two families, All r= chaea studied to date have DNA polymerases in the same family as the replicative eukaryotic DNA polymerases (B family poly- smerases; table 15.2) and are called Pol B. However, members of fone archaeal phylum, Euryarchaeota, have in addition (o Pol Ba ‘unigue archaeal DNA polymerase called Pol D. Pol D enzymes are so distinct, they are the sole members of the D family of DNA polymerases (able 15.2). Current models of DNA replica- tion in euryarchaeotes propose that Pol D has two functions: lagging. strand synthesis and initiation of leading-strand synthe- Figure 15.5 AnArchaea!Repleation Fork. The roplsome poten ad thee unctins are elie table Ts. Compare ths gue to gues 18.18 and 15. sis, Pol B is thought fo eventually replace Pol D and complete synthesis of the leading strand, Retrieve, Infer, Apply 1. Whatis the advantage of having matipe origins ofrepcatin for ‘eukaryote? Whatis the most lousblereason some archaeal species have mulpecrigns? 2. Referto gure 15: and draw the fet replication fork resent near the end ofa chromosome. Use your erawing to ilstrate the end replcaon probe. 4. Review he stepsin toss and mess. Why would having Unprotected ends of near ONA srt these two important cellar processes? 4. Oulne the process use by telomerase to maintain chromosome length. 5. The discovery of telomerase actviyin an archaeon would be 2 suprise. Why? 15.3 Transcription ‘ter reading this section, you should be able to 1 Compare and contrast gone stuelue of eukaryote, archaea bacterial ces 1 ceateatable to stingush eukaryotic archaeal ane bacterial promoters, RNA polymerases, and transcription factors 1 Dscuss the modifications made to eukaryotic mRNA molecules uring and after sythess ‘Transcription is essentially the same in all cellular organisms: the template strand directs RNA synthesis, and RNA is made from the 5” to the 3° end by enzymes called RNA polymerases. Although ‘this unifies the process of transcription aeross all domains af ie, several Key aspects differ. These arise in part from differences in cell and chromosomal structure, the nature of RNA polymerase, and the organization of protein-coding genes. Bacteria use a single [RNA polymerase that consists of the eatalyic core responsible for synthesizing mRNA and the intrinsic transcription factor subunit, sigma (a; se figure 13.23), Bacterial genes with related function axe offen organized into operons, and are thereby transcribed from the same promotes, This results in a polycistonic mRNA (see {figure 13.22) from which individual proteins are translated. Some regions of a bacterial gene (e.g, leader and trailer) are transcribed ‘but not translated, and fr almost all protein-coding genes, the cod- ing information within a gene is continuous. Now let's compare {his to transcription in eukaryotes. Transcription in Eukaryotes In eukaryotes, transeription occurs in the nucleus, and the RNA products must move to the cytoplasm, where they function in translation, Just as noted for DNA replication, chromatin struc- tute must be altered for RNA polymerases to be recruited to promoters and ahead of the RNA polymerases as transcrip- tion proceeds. This is accomplished by chromatin-remodeling and chromatin-modifying enzymes (p. 364). Gene structure and 153 Transcription 385 ‘organization in eukaryotes also is strikingly different from that seen in bacteria. For the vast majority of eukaryotes, each protein- coding gene has its own promotes, and the tanseript synthesized rom the gene is monocistronie. Furthermore, many eukaryotic protein-coding genes are composed of sequences called exons and introns. An exon contains sequences that code for part of the polypeptide, whereas an intron is a stretch of noncoding sequences. Thus the coding portion of a eukaryotic gene is not continuous. Both exons and introns are transcribed, yielding fan eXon- and intron-containing transcript called a primary or pre-mRNA. The introns are spliced out of the primary transcript ‘before the protein is made (p. 356). Eukaryotic RNA Polymerases Include Homologues of the Bacterial RNA Polymerase Core Subunits Bukaryotes have three major RNA polymerases. RNA poly: merase I catalyzes rRNA synthesis, RNA polymerase Ii re- sponsible for mRNA synthesis, and RNA polymerase IIL synthesizes all the tRNAs and one rRNA (5S RNA). Al five ff the bacterial RNA polymerase core enzyme subunits are ‘conserved in eukaryotic RNA polymerases (table 15.3). How- ever, eukaryotic RNA polymerases have several additional subunits, ee ees BacteriaCore RNAP Archaeal RNAP Eukaryotic RNAP I Eukaryotic RNAP Il Eukaryotic RNAP | | subunits Subunits? Subunts Subunits Subunits fe pot pos eo 160 190 | lp p02 (RpoB) 62 ne A135 | ie SE re "ED a | E tot et a fa a | ie ae is = a | sons ont = wes ores | 0010 RpoN) P=10 PaO Rea10 | a ween we I } so en wo cn zm | oe wear cas rs } sy = m= | | RBS cn a2 ] Eukaryotic Transcription Homologous Archaeal | Factor Transcription Factor Taainding proten TSP ree} | ret 18 | | ore we | | rir | ne | | Tn L J Transcription Initiation in Eukaryotes Most research on transcription in eukaryoles has been on the transcription of protein-coding genes, and that is our focus here. In eukaryotes, RNA polymerase II (often referred to as RNAPID) is responsible for transcribing protein-coding genes. This en- zyme is a large aggtegate, atleast 500,000 daltons in size, with about 10 subunits (able 15.3), ‘Transcription initiation requires that RNAPII be recruited to 1 promoter. This is accomplished by transcription factors called basal or general transcription factors, some of which bind the core promoter and then interact with other transcription factors to attract RNAPII to the promoter (table 18.4). RNAPIL-tecognized promoters usually have two regions, the core promoter and a eg- ‘latory region. The core promoter has been defined by in vitro studies to be the minimal region needed for transcription to oc- cur. One of the first features discovered in eukaryotic RNAPIL core promoters was the conserved nucleotide sequence TATA; ‘hus the region is named the TATA box (figure 15.6). Although it was initially thought that all RNAPIE core promoters contain a TATA box, tis now known that many RNAPII core promoters lack a TATA box. TATA-less promoters are generally associated with housekeeping genes. Those that do contain a TATA box tend tobe part of highly regulated genes. Eukaryotic transcription factors are referred to as extrinsic factors because they are not part of the RNA polymerase. The TATA-binding protein (TEP) is one of the most important for ranscription initiation. It is often part of a complex of proteins called TFUD (hort for ranscription factor D for RNA poly- merase IJ). Despite its name, TBP functions in transcription ini- Uistion for both TATA box-containing promoters and those that lack the TATA box. One sequence of events to zectuit RNAPII 0 & (Lore Hoan He Home “art 82 Rte 26 —2t0 14 12810 12 (9) Eukaryotic RNA Polymerase I core promoter ce (are Hoa He 8 (0) Neheeal promoter Figure 15.6 Eukaryotic and Archaeal Promoters. polyrerase core promotes contain one o owver, itis very are fr all ofthe laments tobe is) Euearyove RNA afte elements sro¥n, se BRE the TUB recognition elerentnr isthe inal eleent trenscrson ints within ths ste read bythe aren, OPE she downsteam promater element The approximate postions of e2ch promoter element ae dicated, eave tothe transeipional star ste, An archaea prometer Pomorer element designations ace the same as thse in eukaryeri promeres. the promoter is outlined in figure 15.7. As seen there, initiation requires the formation ofthe pre-initiation complex (PIC). Once formed, TFIIH acts asa helicase to unwind the DNA, forming an ‘open complex (se figure 13.26). RNAPII can then begin synthe- sizing mRNA. However, transcription cannot continue to the elongation phase unl the contacts between TRIB and RNAPIL are broken. This is accomplished by TFIIH, which phosphory- lates the C-terminal domain of one of the RNAPIL subunits (Once this occurs, RNAPIL is released from the PIC and elonga- Lion proceeds. Studies of transcription initiation in numerous eukaryotes ‘have shown that this is not the only sequence of events nor are these the only proteins involved. This is especially (rue for highly regulated genes. For these genes, three protein com- plexes that function as co-activators are important. One is a complex of proteins called mediator. Another co-activator is called SAGA, Both mediator and SAGA are sometimes in- volved in recruiting certain basal tcanscription factors to the promoter during transcription initiation. Interestingly, the basal lranseription factor TPUD is also a co-activator and plays im- portant roles in regulating gene expression, as we discuss in section 15.5. From Pre-mRNA to mRNA: A Unique Eukaryotic Process Unlike nearly all bacterial mRNAs, the initial transcript synthe- sized from a eukaryotic protein-coding gene mast be modified before giving ris to a translatable mRNA. Modification occurs in the nucleus and begins during the elongation phase of transcrip- Lion; i€ is completed after transcription ends. Modification starts With the addition of aa unusual nucleotide to the 5° end of the transcript when the transcript is only about 25 ribonucleotides long (figure 15.8). The 5" eap, as ts called, is 7-methylguano- sine. After the S' cap is added and a sufficiently long transcript is generated, removal of introns begins, Afler synthesis is com- pleted, any remaining introns are removed, and the pre-mRNA is modified further by the addition of a 3° poly-A tail, about 200 nucleotiées long (figure 15.8). Both the 5” cap and poly-A tail help protees the mRNA from enzymatic attack, Further ore, they enable the cell to recognize that the mRNA is intact and ready to be transported from the nucleus to the cytoplasm, ‘The 5’ cap has the additional important function of serving as & recognition signal for binding of ribosomes to the mRNA so that it can be translated Introns are removed from the pre-mRNA by a large complex of proteins and RNA molecules that is unique to ev- Kkaryotic cells. This large complex, called a spliceosome, is composed of several small nuclear ribonucleoproteins (GoRNPS, pronounced “snurps”) and several non-snRNP splicing factors, The sRNPs consist of small nuclear RNA (¢nRNA) molecules bout 60 10 300 nucleotides long) associated With proteins. Some snRNPs recognize and bind exon-intron junctions (figure 15.9). Sometimes a pre-mRNA ie spliced so that dif. ferent patterns of exons remain or the junction of two exons varies. This alternative splicing allows a single gene to code for more than one protein, ‘The splice pattera determines which protein is syn- thesized. Splice patterns can be cell-type specific ‘or determined by the needs of the cell. The impor- tance of alternative splicing in multicellular eu aryotes was clearly demonstrated when it was discovered that the human genome has only about 20,000 genes, rather than the anticipated 100,000. [is thought that alternative splicing is one mecha- nism by which human cells produce a vast array of proteins using fewer genes. This mechanism for ‘expanding the coding capacity of a genome is not available to bacteria and archaea because in their protein-coding genes are very rare. @ Pro cessing of Genetic Information: Prokaryotes Versus Eukaryotes Tae Transcription in Archaea If transcription in archaea were described in a newspaper article, the headline might be some- thing like this: “Eokaryoti-like RNA polymerase functions in bacteriaclike environment.” This is ‘because archaeal transcription occurs in the cytoplasm, as it does for bacteria, Many (although pethaps not quite as many) archaeal genes are oF ‘ganized into operons, as are bacterial genes. Thus ate zany archaeal mRNAs are polyeistronic. Introns are rare in protein-coding archaeal genes (as they are in actera), and they are removed by a process Figure 15.7 Intition of Transerption in Eukarytes. of the preintation comple. Schematic soning the lo ors inthe pe-ntallon complex PI) RNA polymerase is brow Jo proteins of TF. TBPs. proteins of TE, TFB fs yllow. TF and TFA are ot wise i this vew af the how closely the trans shows the direct on 387 “TiAbincing protein TBP) bins the TATA box. This bends the DNA about 9, | TFIA binds DNA upstream of TATA box | ‘TFIB nteraetswtn TBP | BANA pelymerase i and TFF oi the othe proteins atthe promoter TFS irteracs extensively wth RNA polymerase I acting as abridge between "TBP and ANA pelymorae “TEIE then TF bind forming the pre-intation complex {a) Formation ofthe Pre-inition Complex RNA polymerase (6) Precintation Compox Dutlne ofthe formation ion of some athe vanscriovon The two red proteins ne orange snape remresents he two quoi Nite ‘on factors ate associates with RNA, iyerace The arow Sn of vanseription, ance tines. 358 cuanren i | Euksryatic anc Archaeal Genome Replication and Expression T-methylgunosine exp Cap structure at the 5 end of eukaryote ‘mRNA is added soon ater transcription Dosis. 5 we ay bh 5 denne nucleotides added ser tanscision, | ‘A polyA tal consisting of 100-200 AAAAABAR Pol a poly-Atal at the 2 end of eukaryotic mRNA Addition o different than that used by eukaryotes (again as they are in bacteria). Finally, archaeal cells use a single RNA polymerase to catalyze transcription, ‘The archacal RNA polymerase is large and most similar to RNA polymerase II of eukaryotes (table 15:3 and figure 15.10) Likewise, transcription initiation in archaea is very similar to ‘hat seen in eukaryotes as evidenced by the similarity in promot- ers (figure 15.6) and the use of similar basal transcription factors (able 15.4). The archaeal process can be thought of as a simpli- fied version ofthat in eukaryotes. As shown in figure 15.11, 80 archaeal transcription factors are important for reeruiting the archaeal RNA polymerase to a promoter: TATA-binding protein (TBP) and (anseription factor B (TFB). A third transcription factor TFE then associates with RNA polymerase and promotes ‘unwinding of tke DNA to form the open complex (se figure 13.26). RNA polymerase then moves down the gene, transcribing it. ‘TEB remains behind bound to the promoter, ready to initiate another round of transeription. TFE remains with RNA poly- merase until transcription is terminated. Retrieve, Infer, Apply 1. What elements ir raeal promoters ae ako observed in eukaryotic NA polymerase {RNAPI core promoters? What adianal clements are observed in RNAPII cote promters? How de bacterial romersdifer rom these seen in eukaryotic and archaeal ces? 2. Compare the role of bacteria sigma factors in anscipton ination to that of eukaryotic and archaeal transcription factors. Why are bacterial sigma factors refered to a ininscranerption actors, ihereas eukaryotic and archaeal transcription factors ae extnac? 3. Outline the events that generate a mature, translatable mANA\n ‘eukaryotes, Where does this processing occur? Figure 15.8 Medications Made to the Ends of Eukaryotic mRNA. MICRO INQUIRY What urctons ore served by the S* cap ona the 5 pola toi? 15.4 Translation and Protein Maturation and Localization After reading ths section, you should be able to 1 Diterertiate the structure of eukaryote, archaeal and bacterial ribosomes "cent thentator NA used by eukaryotic and archaeal cls 1 Outline the steps in translation ination observed in eukaryotes and compare them tothose observed in bacteria and archaea 1 Discuss the role of molecular chaperones in eukaryote and archaeal 1 reste concept map or abe that distinguishes the trsnsocation and secretion ystems observed in the thee domains of ife For organisms to survive, they must be able to synthesize the proteins they need (translation), fold them into a functional con- formation, and localize the proteins tothe correct site within (or foulside) the cell. In bacteria, translation initiation requires three protein initiation factors (IF), the initiator RNA (fMetRNA,™*), and the two ribosomal subunits (50S and 30S subunits) (see {igure 13.38). The 16S tRNA and the Shine-Dalgarno sequence interact © position the ribosome properly at the start codon. Elongation is assisted by three clongation factors (EFS; see Jigure 13.39), Translation is tightly coupled with transcription, and polysomes are often observed (see figure 13.33). Translation termination involves three release factors (RFs). Chaperones help the protein fold (ge figure 13.43), and some chaperones deliver proteins to protein translocation or sectetion systems. In this section, we focus on translation, and protein folding and localization as they occur in eukaryotes and archaea, As s Pre-maNA 5 slew Ste Bon 1) Furs 2 subunit bind 15" splice ste and Deane site 8! see branche | x snk subunits 2) Additional subunits ‘ind, erating a loop. —— ‘suounte ¥ 3) Stapice ste is ext $'ehd of intron ie onnected othe braneh site. TWo subunits ae released, + a 4) 8 splice site is et [Exon 1s connected Ion plus to cwon 2. The intron Heil {inthe form a loop) released along with the rest of he ssbunite and degaded hee Exon 1 Exon 2 Figure 15.9 Removal of anintron by a Spiceosome. 15.4 Translation anc Protein Maturation anc Localization 359 BBE? Rood-npo? (6) RNA pobymerasell Figure 15.10 Bacterial Archaeal, and Eukaryotic RNA Plymerases Ron diagrams ofthe RNA polymerases af he bectrur Thermus _equocus, the archaea Sulobus state, ane RNA polmerase I ot ‘euaryotes. To subuns fone in alivee RNA polmerases ae snow he. “Those that a unique to achaee and RNA polmorase I ate oan in magenta Stat site Figure 15.11 Binding of TBP and TFB to an Archaeal Promoter. Tho crystal stucture of te teary complex between TB2 the crboxyterminus of TF, anathe region ofan archaeal promoter containing the TATA box and BRE. DNA's stown in gray. Tis the yellow ibbon stucture, and TFBS ‘magenta and turquoise. The turquaise portion of TF8is an ache that recognizes BRE. TaP is TTA-box bd ng protein TFB transcription fair BRE Is Brecogntion element 360. cnarrent | Eukaryotic and Archaeal Genome Replication and Expression ‘Adstional cap MANA, AuG PAB. RARARAAA, oe Figure 15.12 Translation Inationin Eukaryotes Ta simply the drawing, many addonalevearyattiation Factors ls) ae sown Te dope let port ono the figue shows mR aetvalion tne une tight shows the formation ofthe 405 comple. The 405 comalx binds to the end ofthe sctiated mRNA ana then moves cown the MANA torte ts: AUG. This postions the nator "RNA canying methionine what wil Become the pep (P)ste ofthe some ater the adtion ofthe 608. subunit The acceptor) site is vacant ne esd forthe alo he tRNA cartyng the second amino cit encod bye mRNA, Matis methionine, PARP poh binding poten. Este sent ste with DNA replication and transcription, the overall processes are similar in all organisms. Therefore we focus our attention primarily on those aspects of the eukaryotic and archaeal pro- cesses that differ from those observed in bacteria Translation in Eukaryotes Initiates in a Unique Manner but Proceeds Like in Bacteria ‘There axe many obvious differences between bacterial and ev xayotic uansaton. One is that eukaryotic tbosoues ate lager than those in bacteria and require more inition factors to be positioned properly onthe mRNA. The 5’ cap and the 3" poly- ‘Atul of eukaryotic mRNAs ae integral to tanslation int tion, The process begins with mRNA activation. mRNA. activation is brought about by the binding of several eukaryotic ination factors (eI) tothe Send ofthe mRNA and attachment of poly-A binding protein (PABPs) to the 3° end. The elPs and PABPs interact, causing the mRNA to fold back on iselt With the elFs and PABPs forming a bridge between the two ends (figure 15.12), Meanwhile, the initiator (RNA (Met-tRNAS*) is Aug oop Intaton 608 subunit tetors 2 pay n AnAnans se : ware G vs —F oe 485 complex lh rcararan 425. RNA complex Met ave 438 complex seans RNA to fst AUG. Met has ARARAARA Pte, Aste pAnaan a 80S+mANA complex Toaded onto the 40S tibosomal subunit (the small subunit, SSU) to form a structure termed the 43S complex, Formation of the 43S complex is facilitated by several addtional elP, one of whieh de- livers the initiator RNA tothe 40S subunit. The 438 complex then binds the activated mRNA, generating the 43S-mRNA complex. As seen in figure 15.12, at this point in translation initiation, the SSU isnot positioned atthe start codon of the mRNA as itis in ‘actria, Rather, tis atthe S' end of the mRNA. The 438 complex slides down the mRNA seanning for the start codon, Once the start codon is encountered, scanning ceases and the 60S ribosomal ‘subunit large subunid joins with the 43S-mRNA complex, form- ing the complete 80S ribosome. Translation can now commence. Our description of translation initiation holds for mRNAs having a leader (i., a5" untranslated region). In recent years mRNAs lacking a leader have been identified, For instance, all mRNAs produced by the protist Giardia intestinalis lack lead- cers, How these leadesless mRNAs are translated is still uncleat However, evidence suggests that intact 80S ribosomes bind the start codon and initiate translation, ‘Translation elongation and termination proceed in a fash- ion similar to that seen in bacteria. Thice elongation factors function in both bacteria and eukaryotes, and the bacterial and ‘eukaryotic factors are very similar structurally and function- ally. Polysomes are used during translation of eukaryotic mRNAs. However, because of the circularization of the mRNA

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