40632_CH08_151_188.qxp 12/14/06 12:12 PM Page 151 8 Protein Synthesis CHAPTER OUTLINE 8.1 Introduction • The rRNA of the 30S bacterial ribosomal subunit has a com- plementary sequence that base pairs with the Shine– 8.2 Protein Synthesis Occurs by Initiation, Elongation, Dalgarno sequence during initiation. and Termination 8.8 Small Subunits Scan for Initiation Sites on Eukaryotic • The ribosome has three tRNA-binding sites. mRNA • An aminoacyl-tRNA enters the A site. • Eukaryotic 40S ribosomal subunits bind to the 5′ end of • Peptidyl-tRNA is bound in the P site. mRNA and scan the mRNA until they reach an initiation site. • Deacylated tRNA exits via the E site. • A eukaryotic initiation site consists of a ten-nucleotide • An amino acid is added to the polypeptide chain by trans- sequence that includes an AUG codon. ferring the polypeptide from peptidyl-tRNA in the P site to • 60S ribosomal subunits join the complex at the initiation aminoacyl-tRNA in the A site. site. 8.3 Special Mechanisms Control the Accuracy of Protein 8.9 Eukaryotes Use a Complex of Many Initiation Factors Synthesis • Initiation factors are required for all stages of initiation, • The accuracy of protein synthesis is controlled by specific including binding the initiator tRNA, 40S subunit attach- mechanisms at each stage. ment to mRNA, movement along the mRNA, and joining of 8.4 Initiation in Bacteria Needs 30S Subunits and Accessory the 60S subunit. Factors • Eukaryotic initiator tRNA is a Met-tRNA that is different from the Met-tRNA used in elongation, but the methionine • Initiation of protein synthesis requires separate 30S and is not formulated. 50S ribosome subunits. • eIF2 binds the initiator Met-tRNAi and GTP, and the complex • Initiation factors (IF-1, -2, and -3), which bind to 30S sub- binds to the 40S subunit before it associates with mRNA. units, are also required. 8.10 Elongation Factor Tu Loads Aminoacyl-tRNA • A 30S subunit carrying initiation factors binds to an initia- tion site on mRNA to form an initiation complex. into the A Site • IF-3 must be released to allow 50S subunits to join the • EF-Tu is a monomeric G protein whose active form (bound to 30S-mRNA complex. GTP) binds aminoacyl-tRNA. 8.5 A Special Initiator tRNA Starts the Polypeptide Chain • The EF-Tu-GTP-aminoacyl-tRNA complex binds to the ribo- some A site. • Protein synthesis starts with a methionine amino acid usu- ally coded by AUG. 8.11 The Polypeptide Chain Is Transferred to Aminoacyl-tRNA • Different methionine tRNAs are involved in initiation and • The 50S subunit has peptidyl transferase activity. elongation. • The nascent polypeptide chain is transferred from peptidyl- • The initiator tRNA has unique structural features that dis- tRNA in the P site to aminoacyl-tRNA in the A site. tinguish it from all other tRNAs. • Peptide bond synthesis generates deacylated tRNA in the P site and peptidyl-tRNA in the A site. • The NH2 group of the methionine bound to bacterial initia- tor tRNA is formylated. 8.12 Translocation Moves the Ribosome 8.6 Use of fMet-tRNAf Is Controlled by IF-2 and the Ribosome • Ribosomal translocation moves the mRNA through the ribo- some by three bases. • IF-2 binds the initiator fMet-tRNAf and allows it to enter the partial P site on the 30S subunit. • Translocation moves deacylated tRNA into the E site and 8.7 Initiation Involves Base Pairing Between mRNA and rRNA peptidyl-tRNA into the P site, and empties the A site. • The hybrid state model proposes that translocation occurs • An initiation site on bacterial mRNA consists of the AUG ini- in two stages, in which the 50S moves relative to the 30S, tiation codon preceded with a gap of ∼10 bases by the and then the 30S moves along mRNA to restore the original Shine–Dalgarno polypurine hexamer. conformation. 151 40632_CH08_151_188.qxp 12/14/06 12:12 PM Page 152 8.13 Elongation Factors Bind Alternately • Virtually all ribosomal proteins are in con- tact with rRNA. to the Ribosome • Most of the contacts between ribosomal • Translocation requires EF-G, whose struc- subunits are made between the 16S and ture resembles the aminoacyl-tRNA-EF-Tu- 23S rRNAs. GTP complex. 8.17 • Binding of EF-Tu and EF-G to the ribosome Ribosomes Have Several Active Centers is mutually exclusive. • Interactions involving rRNA are a key part • Translocation requires GTP hydrolysis, of ribosome function. which triggers a change in EF-G, which in • The environment of the tRNA-binding sites turn triggers a change in ribosome is largely determined by rRNA. structure. 8.18 16S rRNA Plays an Active Role in Protein 8.14 Three Codons Terminate Protein Synthesis Synthesis • 16S rRNA plays an active role in the func- • The codons UAA (ochre), UAG (amber), tions of the 30S subunit. It interacts and UGA terminate protein synthesis. directly with mRNA, with the 50S subunit, • In bacteria they are used most often with and with the anticodons of tRNAs in the P relative frequencies UAA>UGA>UAG. and A sites. 8.15 Termination Codons Are Recognized 8.19 23S rRNA Has Peptidyl Transferase by Protein Factors Activity • Termination codons are recognized • Peptidyl transferase activity resides exclu- by protein release factors, not by sively in the 23S rRNA. aminoacyl-tRNAs. 8.20 Ribosomal Structures Change When the • The structures of the class 1 release fac- Subunits Come Together tors resemble aminoacyl-tRNA-EF-Tu and • The head of the 30S subunit swivels EF-G. around the neck when complete ribosomes • The class 1 release factors respond to spe- are formed. cific termination codons and hydrolyze the • The peptidyl transferase active site of the polypeptide-tRNA linkage. 50S subunit is more active in complete • The class 1 release factors are assisted by ribosomes than in individual 50S subunits. class 2 release factors that depend on GTP. • The interface between the 30S and 50S • The mechanism is similar in bacteria subunits is very rich in solvent contacts. (which have two types of class 1 release 8.21 factors) and eukaryotes (which have only Summary one class 1 release factor). 8.16 Ribosomal RNA Pervades Both Ribosomal Subunits • Each rRNA has several distinct domains that fold independently. 8.1 FIGURE 8.1 shows the relative dimensions of Introduction the components of the protein synthetic appa- An mRNA contains a series of codons that inter- ratus. The ribosome consists of two subunits that act with the anticodons of aminoacyl-tRNAs so have specific roles in protein synthesis. Messen- that a corresponding series of amino acids is ger RNA is associated with the small subunit; incorporated into a polypeptide chain. The ribo- ∼30 bases of the mRNA are bound at any time. some provides the environment for controlling The mRNA threads its way along the surface the interaction between mRNA and aminoacyl- close to the junction of the subunits. Two tRNA tRNA. The ribosome behaves like a small migrat- molecules are active in protein synthesis at any ing factory that travels along the template moment, so polypeptide elongation involves engaging in rapid cycles of peptide bond synthe- reactions taking place at just two of the (roughly) sis. Aminoacyl-tRNAs shoot in and out of the ten codons covered by the ribosome. The two particle at a fearsome rate while depositing tRNAs are inserted into internal sites that stretch amino acids, and elongation factors cyclically across the subunits. A third tRNA may remain associate with and dissociate from the ribosome. on the ribosome after it has been used in pro- Together with its accessory factors, the ribo- tein synthesis before being recycled. some provides the full range of activities The basic form of the ribosome has been required for all the steps of protein synthesis. conserved in evolution, but there are apprecia- 152 CHAPTER 8 Protein Synthesis 40632_CH08_151_188.qxp 12/14/06 12:12 PM Page 153 ble variations in the overall size and propor- A ribosome binds mRNA and tRNAs tions of RNA and protein in the ribosomes of bacteria, eukaryotic cytoplasm, and organelles. FIGURE 8.2 compares the components of bacte- rial and mammalian ribosomes. Both are ribonucleoprotein particles that contain more 200 Å 60 Å RNA than protein. The ribosomal proteins are known as r-proteins. Each of the ribosome subunits contains a 60 Å major rRNA and a number of small proteins. 220 Å The large subunit may also contain smaller RNA(s). In E. coli, the small (30S) subunit con- 35-base mRNA sists of the 16S rRNA and 21 r-proteins. The large (50S) subunit contains 23S rRNA, the FIGURE 8.1 Size comparisons show that the ribosome is small 5S RNA, and 31 proteins. With the excep- large enough to bind tRNAs and mRNA. tion of one protein present at four copies per ribosome, there is one copy of each protein. The major RNAs constitute the major part of the Ribosomes are ribonucleoprotein particles mass of the bacterial ribosome. Their presence is pervasive, and probably most or all of the Ribosomes rRNAs r-proteins ribosomal proteins actually contact rRNA. So 23S = 2904 bases 31 Bacterial (70S) 50S the major rRNAs form what is sometimes 5S = 120 bases mass: 2.5 MDa thought of as the backbone of each subunit—a 66% RNA 30S 16S = 1542 bases 21 continuous thread whose presence dominates the structure and which determines the posi- 28S = 4718 bases 49 tions of the ribosomal proteins. Mammalian (80S) 60S 5.8S = 160 bases The ribosomes of higher eukaryotic cyto- mass: 4.2 MDa 5S = 120 bases plasm are larger than those of bacteria. The total 60% RNA 40S 18S = 1874 bases 33 content of both RNA and protein is greater; the major RNA molecules are longer (called 18S FIGURE 8.2 Ribosomes are large ribonucleoprotein parti- and 28S rRNAs), and there are more proteins.