or 18S, 58S, and 28S TRNAS are foun ec Meeaes wpicaly tandemly repeated 100 te aac Nie depending on the organism), to form one oq foe fl the fepeat nits, a nucleolus forms aroweal oe cr Typically the muluple nucleoli so formed fuses se nucleolus 0 lust tach eukaryotic DNA repeat unit is transcribed NA polymerase I to produce a pre-rRNA molec . ‘ule wit the organization 5'-18S-5.85-285-3', which has pe Cequences between each FRNA and at the 5” and 5! nds. Processing by specific ribonucleases generates the thie «RNAS by removing the spacers. The pre-tRNA. processing events take place in complexes formed be, tween the pre-FRNA, 5S rRNA, and ribosomal proteins The 55 FRNA is produced by transcription of the 5S URNA genes (typically located elsewhere in the genome) by RNA polymerase IIL. As pre-tRNA processing pro- ceeds, the complexes undergo changes in shape, restlt- ing in formation of the functional 60S and 40S ribosomal subunits, which are then transported to the cytosol Ic is important to be clear about the distinction be- tween an intron and a spacer. The removal of a spacer releases the flanking RNAS, and they remain separate. In- on removal, by contrast, results in the splicing together of the RNA sequences that flanked the intron. Keynote Ribosomes consist of two unequally sized subunits, each containing one or more ribosomal RNA molecules and ribosomal proteins. The three prokaryotic rRNAs and three of the four eukaryotic RNAs are encoded in rRNA transcription units. The fourth eukaryotic NA is en- coded by separate genes. The transcription of rRNA tran- scription units by RNA polymerase produces pre-rRNA ‘molecules that are processed to mature rRNAS by the re- ‘moval of spacer sequences. The processing events occur ‘in complexes of the pre-rRNAs with ribosomal proteins and other proteins and are part of the formation of the ‘mature ribosomal subunits. # Initiation of Translation The three basic stages of protein synthesis—initiation, ongation, and termination—are similar in bacteria and eukaryotes. In this section and the two sections tat tok imatic low, we discuss each of these stage Mimation — i, wun, concentrating on the Intation of processes in E. coli. In the discus- Translation Sions, significant differences ,in translation between bacteria and cukaryotes are noted. 7 Initiation encompasses alLaL hic step> preceding the fon en the first 0 Bitton of the peptide bend between the first wo 3mIn sn he PolYPEPide chain. Initiation involves wlecule, a ribosome, a specif “RNA, Prot ekg fbOSOME, # spec innor RNA, phosphate) and GTP {Initiation in Bacteria. in ba Caton of aren 8 bacteria, the first step in the in ribosomal subunit to aids in the binding ofthe sub Uunit to mRNA and prevents bindin, subunit to the 305 Labonte binding of the 505 ribosomal ‘The AUG initiation codon alone is not sufficient to in dicate where the 305 subunit should binid to the mRNA. a Sequence upstream (to the 5' side in the leader of the MRNA) of the AUG called the ribosomé-binding, site (RBS) is also needed: In the 1970s, John Shine and iynn Dalgamo hypothesized that the purine-tich RBS sequence (5'-AGGAG-3" or some similar sequence) and sometimes other nucleotides in this region could pair with a comple- mentary pyrimnidine-rich region (always containing the #- ‘quence 5'-UCCUGG= 3’ at the 3° end of 16S FRNA (Figure 6.16). Joan Sjeitz was the frst to demonstrate this pairing experimentally. The mRNA RBS region is now c known as the Shine-Dalgarno’ sequence. ‘Most of the RBSS ate 8 T0 12 nucleotides upstream from the initiation codon. The models that the forma- tion of complémentary base pairs between the mRNg and 165 rRNA allows the stall ribosomal subunit to locate'the true sequence in the TRNA for the initiation of protein sfnthesis-Genetic evidence sipports this model. If the Shine-Dalgarno sequence of an mRNA is mutated so that its possible pairing with the 165 tRNA sequence is significantly diminished or prevented, the mutated mRNA cannot be translated. Likewise, if the rRNA se- quence complenientary to the Shine-Dalgarno sequence is mutated, mRNA tranBlation cinnot occur. Since it can be argited that the loss of translatability’as a résult of mu tations in one or the other RNA partner could be caused by effects unrelated to the loss of pairing of the two RNA segments, a’mor¢ elegaiit experiment was done. That is, mutations were made in the Shine-Dalgarno sequence to abolish pairing with the wild-type tRNA sequence, and compensating mutations were made in the rRNA se quence so that the two mutated sequences cauld pair. In this case, mRNA translation occurred normally, indicat ing the importance of the pairing of the two RNA seg ments, (This type of experiment, in which compensating ‘mutations are made in two sequences that are hypothe- sized to interact, has been used in a number of other systems to explore the roles of specific interactions in biological functions.) i “Fhe next step in the initiation of translation 1 the binding of a special initiator URNA to the AUG start codon to which the 305 subunit is bound. In both prokaryotes sing eukaryotes, the AUG initiator codon specifics me thicnine, As a result, uewly made proteins in both types mmonly 115 nig au wonessued, sisayukeA116 al subunit Shine-Dalgarno 90S ribosom: Eeauares pada ¢ , I tolled AY. Clie. 90S. ribosomal subunit mANA S'bebntababed . binds to mRNA 2 ¢ 3 = maNA 2 $s 3 Wit tRNA Binds to BJ — met initiator tRNA MRNA complex GTP—Q) Ser —iF-2 WAC 708 iitason Of organisms beo;.ya 6.16 __ Peace involved in the 18 of ribosomes to the mRNA ‘he intiaion of protein synthesis in prokaryotes, ) Sequence at 3" end of 165 FRNA AUUCCUCCAUAG™ = 5 1) Example of sequence upstream of the AUG codon in an mRNA pairing with the 3° end of 16S rRNA Shine-Dalgarno sequence Initiation ‘codon | 5 UGUACUA AGGAGGUUG UG)GAACAACac / “165 (RNA 3 end 3 | the resulting molecule is designated IMet-tRNA {Met \ (this nomenclature indicates that the tRNA is specific for the attachment of fMet and that (Met ‘is attached toi.) = . . . Note that, when an AUG codon in an mRNA mole- cule is encountered at a position other than the start of the amino acid-coding sequence, a different tRNA, called IRNA.Met, is used to insert methionine at that point in the polypeptide chain. This tRNA is charged by the same aminoacyl-tRNA synthetase as is tRNA.fMet to produce Met-tRNA.Met. However, tRNA‘Met and fet molecules are coded for by differeit geries and have dif- ferent sequences. We'will see later in the chapter how the two tRNAs are used differently. - The initiator tRNA, fMet-tRNA.(Met, is brought to the 30S subunit-mRNA complex by IF-2, which also car- fies a molecule of GTP. The initiator tRNA binds to the subunit in the P site. We will see later that, subsequently, all aminoacyl-tRNAs that come tothe ribosome bind to the A site. Howevér, IF-1 bound to the 305 subunit is blocking the A site so that only the P site is available for the initiator tRNA to bind to. Formed at this point is the 30S initiation complex, consisting of the mRNA, 30S sub- ‘nit, initiator (RNA, and the initiation factors (see Figure 6.15). Next, the 50S ribosoy it binds, leading to GTP hydrolysis ect he schooner three initiation fac- ‘ots. The final complex is called the 70S initiation com- Plex (sce Figure 6.15). Inatlon In Eukaryotes. The initiation of translation is “ilar in eukaryotes, although the process is more com- Plex and involves Many more initiation factors, called (kent initiation factors (eIF), than is the case in bac- a The main differences are that: (1) the initiator me- suit pat 'S unmodified, although a special initiator tRNA scp 8S Ht the ribosome; and (2) Shine-Dalgarno s« HLL! at not found in eukaryotic mRNAS. Instead, the “HC ribosome uses another way to find the AUS o Initiation codon, First, « eukaryotic initatoy facto el Ab ‘a.multimer of several proteins, including e|F-A1:, the sap binding protein (CBP}—binds ta, the cap at the 9! end nl the mRNA (see Chapter 5), Then, a complex of she 40" ibosomal subunit with the initiator, Melia, sevetal elf pioteins,.and GP binds, together with «ther el and moves along the mRNA. scanning for the \wptyaur AUG codvin. The AUG codon 1s embedded in « sequenice—called the Kozak sequence alter Ma Kozak—which indicates that it is ‘the imitator codon This process is calléd the scanning model for iwityauon The AUG codon, is almost always the first AUG codon from the 5’ end of the codon, jt must be ina Once the 40§ subunit fings this AYG, it binds.te- 1 and then the 605 ribosomal subipit binds, displacing the els Cexcept for elF-4F, which 1s needed for the subsequent initiation of translation), producing the 805’ intauon complex: with the’ initiator Met-tRNA bound to the mRNA in the P site of the ribosome * The poly(A) tail of-the eukaryotic mRNA also plays « role in translation, Poly(A) binding protein 11 (PABPIL, see Figure 5.11b, p. 92) bound to the poly(A) tail also binds to elF-4G, one of the proteins of el'-4¥ at the cap, thereby looping the 3/ end of the mRNA close to the 5° end, In this way, the poly(A) tail stimulates the i of translation, 7 Elongation of the Polypeptide Chain Alter initiation is complete, the next stage is elongation. Figure 6.17 de- @Qnimation picts the elongation events—the ad- Elongation dition of amino acids to the growing of the polypeptide chain one by one—as Polypeptide they take place in bactena This Chain phase has three steps: AC AminoacyltRNA (charged URNA) binds wo the abe me in the A site 2. A peptide bond forms The ribosome moves (tsanslocates) along th one codon. . . ‘As wath initiation, elongation requires accessory prptein factors,-here called elongation factors (LE), and GIP Elongation is similar in eukaryotes - -_ Binding of Aminoacyl-tRNA. At the start of el the anticodon of {Met-tRNA is hydrogen bonded AUG initiation codon in the P site of the boson (Figure 6.17, step 1). The next codon in the mlNA 1s in the A site; in Figure 6.17, this codon (UCC) specifies the (Ser) ppropnate ansinoacyl-tRNA (here ds, the codon im the A» yacy!-tRNA t5 brougl v7 amino acid sed Next, the IRNA Ser) bi Ciyue 617, complex of the protem clon a7 aq woes, ssaquig uleadig 90 SAN| uone|suely :uo|ssaid ava 9 s0\deyg ‘Elongation stage of translation in bact unstable, while the “s* stands for stable Peptidy tRNA ibosome movement 1RNA Sosevent ea, and uncharged IRN separate In ici factor (RRE) and EF-G: Pste S1op codon is encountered J C. Ltt Li fab AAGUAG i) 1, re — mRNA. Stop Release Codon ae Release factor (RF3) binds to stop codon ¢ © Poiypeptide chain isteleased - © AF3-coP bings, re pe fo GTP replaées the a 28 © Aiosome recycling factor (RAF) binds to Asite ribosome ot GTP to GOP causes translocation ofthe ribosome, Bi . ) AFS-GOP | / Oe eS @