Protein synthesis occurs as a result of the formation of peptide bonds formed in ribosomes between amino acids whose sequence is determined by the genetic code carried in mRNA. The genetic code is specified in units of three bases (called a codon). Each codon specifies one amino acid. The essential translator function between nucleic acid sequence and amino acid sequence is provided by tRNAs. Interestingly, the actual reaction where peptide bonds are formed is catalyzed by rRNA, making the ribosome essentially a ribozyme (catalytic RNA) complex. There is only RNA at the site where peptide bonds are formed. The error rate of protein synthesis is consistent with the average length of polypeptides. An error rate of 10-3 means about one error per ten thousand amino acids. Since almost all polypeptides are smaller than this, it means that on average, most polypeptides have no errors (though this is not absolutely true, of course). Higher accuracy is not necessarily desirable, because it may require additional time.
E. coli has 86 tRNAs. Note that the anticodon contains a sequence that forms complementary base pairing with the three base codon sequence in the mRNA during translation. All known tRNAs have between 73 and 93 ribonucleotides. About 7-15 bases per molecule are modified. About half of the bases are involved in intramolecular base pairs in the molecule. Five groups of bases are not in duplexes - 1) the terminal CCA sequence to which the amino acid is attached; 2) the pseudouridine loop, 3) the extra arm, 4) the DHU loop (DHU = dihydrouracil), and 5) the anticodon loop. The 5' end of the tRNA has a phosphate and usually contains a G residue. The tRNA molecule is actually L-shaped in 3D. Duplex regioins of the tRNA are in the 'A' configuration. Non-Watson-Crick base pairs form for some of the bases in the molecule. The four helices in the secondary structure of tRNA stack to from an L-shaped molecule.
The amino acid on the tRNA is covalently attached at the 3' end by enzymes known as aminoacyl-tRNA synthetases. Note that the carboxyl group of the amino acid has formed an ester bond with the 3' hydroxyl of the ribose. Some tRNAs have the same bond on the 2' hydroxyl of ribose. After a tRNA has been linked to its amino acid, it is described as either an aminoacyl-tRNA or a charged tRNA. E. coli has 20 aminoacyl-tRNA synthetases, one for each amino acid. Each aminoacyl-tRNA synthetase recognizes one amino acid and one or more tRNAs to which that amino acid can be attached according to the genetic code (cognate tRNAs).
The first step in aminoacyl-tRNA synthetase catalysis - charging a tRNA with an amino acid - involves the formation of an aminoacyl AMP intermediate from ATP and an amino acid. Next, the aminoacyl group is transferred to a tRNA to form aminoacyl-tRNA + AMP. Thus, the overall reaction uses two high energy phosphates from ATP (forming AMP) to create aminoacyl-tRNA. Pyrophoshate is released in the overall reaction, helping to pull it forward.
Aminoacyl-tRNA synthetases incorporate an incorrect amino acid into a tRNA at a rate of about one per 10,000-100,000 reactions. This reasonably low error frequency is obtained by two types of proofreading: one error correcting mechanism at the level of forming the aminoacyl adenylate intermediate, and the other error correcting mechanism at the level of release of the aminoacylated tRNA itself. If an incorrect amino acid is placed onto a tRNA (for example, if serine were put onto a tRNA that specified threonine), then the incorrect amino acid would be inserted into the protein in place of the correct one if nothing is done. This indicates that the information in the tRNA in the anti-codon is all that is used to place an amino acid into a protein. However, if one takes the tRNA with serine on it which should have had threonine (abbreviated here and in book as Ser-tRNAThr) and incubates it with the threonyl-tRNA synthetase, the serine is rapidly removed from the tRNA. Incubation of Thr-tRNAThr with the same enzyme has no effect, indicating that the synthetase has a proofreading type function on it to edit tRNAs and insure that they have the proper amino acid on them. Two sites of an aminoacid tRNA synthetase are relevant for insuring that the proper amino acid is placed on a tRNA - an acylation site and an editing site. Acylation sites reject amino acids that are too large, whereas editing sites hydrolyze amino acids that are smaller than the correct one. The CCA end of the tRNA is flexible, allowing the end with the tRNA to flip between different sites of the synthetase.
Aminoacyl tRNA synthetases are ultimately the source of the genetic code because they must recognize a tRNA and put the appropriate amino acid onto it. The threonyl-tRNA synthetase forms a complex with the tRNAThr that involves recognition by the enzyme of both the anticodon loop and the 3' CCA sequence of the tRNA. Again, note the interaction of the enzyme with both the 3' CCA sequence and the anticodon loop. These two synthetases are members of the two different classes of aminoacyl-tRNA synthetases. Glutaminyl-tRNA synthetase is a member of Class I and threonyl-tRNA synthetase is a member of Class II. These different classes of enzymes bind to the tRNA molecules in different ways. Class I enzymes put the amino acid onto the 2' hydroxyl of the terminal adenosine of the tRNA, whereas Class II enzymes put the amino acid onto the 3' hydroxyl of the terminal adenosine of the tRNA.
Ribosomes are the protein-rRNA complexes where the process of translation is carried out. Ribosome sizes are measured in Svedberg sedimentation units (for example, the intact E. coli ribosome has a size of 70S). The larger the number, the larger the unit. The relationships between numbers and sizes is not linear or additive, however. The 70S E. coli ribosome is composed of a 50S large subunit and a 30S small subunit. The 30S subunit contains 21 proteins (numbered S1 to S21) and one rRNA molecule with a size of 16S. The 50S subunit contains 34 different proteins (numbered L1 to L34) plus two rRNA molecules with sizes of 23S and 5S. All rRNAs and proteins are present in one copy each, except for proteins L7 and L12, which are present in two copies each. In addition, L7 is identical to L12 except that its amino terminus is acetylated. S20 and L26 are identical. Interestingly, the ribosome can self assemble simply by mixing the proteins and rRNAs together.
Two thirds of the mass of the ribosome is contained in the rRNAs of the complex. rRNAs have extensive intramolecular base pairing and perform important roles in ribosome function, include catalysis of formation of peptide bonds. Proteins in ribosomes provide structural support. Many have elongated structures that reach into the RNA matrix.
Protein synthesis occurs (like DNA synthesis and RNA synthesis) solely in the 5' to 3' direction along a messenger RNA. This allows translation to begin in E. coli before transcription is even completed. If protein synthesis occurred in the 3' to 5' direction, this would not be possible, as the 3' end would not be released until transcription was complete. Translation does NOT begin at the 5' terminus of the mRNA. Rather it starts at the sequence AUG. In some cases, it doesn't even start at the first AUG sequence. How then does the ribosome know which AUG to start at? In E. coli, the proper AUG sequence (called a codon) is identified by virtue of the fact that a common sequence (called a Shine-Dalgarno sequence) is located close to the AUG start codon. The most common version of this sequence is GGAGG, which is complementary to the CCUCC sequence in the 16S rRNA. Thus, base pairing of the Shine-Dalgarno sequence with the 16S rRNA of the ribosome positions the ribosome to begin protein synthesis. Positioning of the ribosome is particularly important in E. coli, because prokaryotes often have multiple related genes coded in an operon present on a single mRNA. Ribosomes must be positioned appropriately for each gene on the mRNA for translation to occur properly.
The start codon AUG codes for the amino acid methionine. This amino acid is used to start synthesis of both prokaryotic and eukaryotic proteins. In prokaryotes, the very first methionine put into a protein (but not subsequent ones) is chemically altered. This involves putting a formyl group onto the amino acid after it is on the tRNA. Interestingly, there are two different tRNAs with anticodons complementary to the AUG start codon that get acylated with methionine. One of these, known as the initiator tRNA, is the one that gets a methionine that gets formylated. The other, simply gets methionine and does not get formylated. The first one is used only to start protein synthesis. The second one is used only to put methionine into proteins AFTER the first one has been used to start the protein.
tRNA Binding and Translation Elongation
tRNAs, of course, bind to ribosomes during the process of translation and pair with the mRNA held by the 30S subunit of the ribosome. The ribosome has three binding sites for tRNA, called the A (aminoacyl), P (peptidyl), and E (exit) sites. Anticodons of the tRNAs in the A and P site form base pair interactions with the mRNA, whereas the one in the E site does not. The end of the tRNA opposite the anticodon interacts with the 50S subunit. The acceptor stems of the A and P tRNAs converge at the site where the peptide bond is formed. A tunnel at this site allows the growing polypeptide to exit through the ribosome during synthesis. The cycle begins with a peptidyl tRNA in the P site. Next, an aminoacyl-tRNA complementary to the next 3 base codon of the mRNA binds in the A site. A peptide bond is then formed at the ribosomal peptidyl transferase center, with the peptide bound to the tRNA in the A site. Then translocation occurs (requires GTP and a protein factor called elongation factor G), with the tRNA in the P site moving to the E site and the tRNA with the peptide on it moving from the A site to the P site and the mRNA moving 3 bases further through the ribosome. The system is then ready to bind another tRNA in the A site. Without the formyl group, the free terminal amino group can cyclize to cleave itself from the tRNA, forming a six member ring and terminating translation. Formylation prevents this from happening.
Binding of tRNAs to the mRNA during translation occurs through interactions between the tRNA anticodon and the codon of the mRNA. Interestingly, some anticodons can recognize more than one codon. It turns out that the third base of a codon is less important for identifying the correct anticodon than the first two. This variable pairing of the third base of the codon with the anticodon is referred to as the wobbly hypothesis. Note that inosine (shown by 'I' in the table) is a possible base in an anticodon due to base modifications that occur in tRNAs. Inosine is the most flexible base for pairing in the tRNA. It is capable of pairing with U, C, or A. Note also that in the genetic code itself, the third base of the codon is often variable. For example, the genetic code for proline can be either CAU, CAC, CAA, or CAG. Thus, in the code itself, the first two bases are the most important and the third base often varies.
For translation to begin, the mRNA and formylmethionyl-tRNA must be brought to the ribosome. Three proteins called initiation factors (IF1, IF2, and IF3) are essential. IF1 and IF3 bind first to the 30S subunit of the ribosome. These serve to prevent the 50S subunit from binding prematurely. IF2 (a G protein) binds GTP, changing its conformation and enabling it to associate with the formylmethionyl-tRNA. This IF2-GTP-formylmethionyl-tRNA complex binds to the mRNA on the 30S ribosome. The mRNA has, by this point, formed base pairs between its Shine-Dalgarno sequence and the complementary sequence at the 3' end of the 16S rRNA of the 30S subunit. The 50S subunit comes and binds as the GTP on IF2 is hydrolyzed and IF2 and IF1 are released. This forms a 70S initiation complex. Note that the initiator tRNA occupies the P site with the A and E sites empty. The ribosomal complex is now ready for elongation, as described partly above.
Elongation (additional details)
For elongation to occur, aminoacyl-tRNAs must be brought to the A site of the ribosome complex. Delivery of aminoacyl-tRNAs occurs through action of elongation factor Tu. EF-Tu is a G protein that binds aminoacyl-tRNA only when it (EF-Tu) contains GTP. EF-Tu serves to protect the ester link in the aminoacyl-tRNA and, interestingly, insure that the proper tRNA is inserted into the A site. EF-Tu performs this latter function by hydrolyzing GTP only when proper pairing between the anticodon of the tRNA and the codon of the mRNA in the ribosome occurs. If proper pairing does not occur, GTP remains bound to EF-Tu and the aminoacyl-tRNA is not released to the ribosome. After EF-Tu transfers the proper aminoacyl-tRNA to the ribosome, EF-Tu is released bound to GDP. GTP is reloaded onto EF-Tu by virtue of assocation with EF-Ts, which unloads the GDP from EF-Tu, allowing GTP to bind, and EF-Ts is released in the process. Note that EF-Tu does NOT bind to the initiator tRNA (formylmethionyl-tRNA), but does bind to the tRNA with regular methionine. This insures then that formylmethionine is not incorporated into protein after the first amino acid.
After the appropriate tRNAs are in the P and A sites, the peptide bond forms spontaneously. For translation to continue translocation of the mRNA and tRNAs on the ribosome must occur. This is mediated by another elongation factor called elongation factor G (EF-G - also called translocase). The structure of EF-G closely resembles that of the complex between EF-Tu and tRNA. This is an example of molecular mimicry - a protein domain mimics the shape of a tRNA. First EF-G with GTP attached, binds to the EF-Tu-like domain of the 50S subunit. The tRNA-like domain of EF-G binds to the 30S subunit. This causes the peptidyl-tRNA in the A site to move one codon down to the P site, carrying the mRNA and the deacylated tRNA previously in the P site with it (to the E site). GTP is hydrolyzed in EF-G in this process, causing EF-G to dissociate, leaving the A site open for the next aminoacyl-tRNA.
Translation termination occurs when one of three special codons called stop codons appears in the A site of the ribosome. The stop codons are UAA, UGA, and UAG. Note that no tRNAs have anticodons complementary to these sequences. Termination of translation is facilitated by binding of proteins called release factors to the stop codon. Release factor 1 (RF1) recognizes UAA or UAG. Release factor 2 (RF2) recognizes UAA or UGA. A third release factor (release factor 3 - RF3) is a G protein homologous to EF-Tu and serves to mediate interactions between RF1 or RF2 and the ribosome. Release factors appear to work by carrying a molecule of water to the peptide bond formation region of the ribosome. The water, thus delivered, acts to hydrolyze the ester bond between the polypeptide on the 3' end of the tRNA in the P site, releasing the polypeptide from the tRNA (and from the ribosome as well). Release of the tRNA and mRNA from the ribosome is assisted by ribosome release factor (RRF) and EF-G. RRF also resembles tRNA.
Eukaryotic Translation Initiation
Translation in eukaryotic and archaean cells is similar to the same process in bacteria. Differences in eukaryotic translation are as follows:
1. Eukaryotic ribosomes are 80S vs 70S in prokaryotes. The 80S complex contains a 60S large subunit and a 40S small subunit. The 40S subunit contains an 18S rRNA homologous to the 16S rRNA of the 30S subunit in prokaryotes. The 60S subunit contains three rRNAs - a 5S rRNA (homologous to the prokaryotic 5S), a 28S rRNA (homologous to the prokaryotic 23S) and a unique 5.8S rRNA.
2. Eukaryotes use methionine instead of formylmethionine as the first amino acid in translation. A special tRNA carrying methionine is used, however, that is different from the tRNA carrying methionine for non-initiation. This is similar to the prokaryotic system.
3. Eukaryotes do not have a Shine-Dalgarno sequence for identifying the start codon (AUG, as in prokaryotes), but usually use the first AUG that occurs closest to the 5' end. The 40S ribosomal subunit binds to the cap of the eukaryotic mRNA (5' end) and sequentially slides down the mRNA (assisted by helicases and ATP energy) looking for the first AUG.
4. Eukaryotes use more initiation factors than prokaryotes. eIF designates a eukaryotic initation factor. eIF-4e binds the cap. EIF-4A is a helicase.
5. Eukaryotic elongation is driven by eukaryotic elongation factors EF1alpha and EF1beta-gamma, which are counterparts to EF-Tu and EF-Ts. EF1alpha bound to GTP delivers aminoacyl-tRNA to the A site of the ribosome and EF1beta-gamma catalyzes the release of GDP and addition of GTP to EF1alpha. Eukaryotic EF2 mediates translation using GTP energy similar to the mechanism of EF-G.
6. A single termination factor called eRF1 performs the functions of RF1 and RF2 of prokaryotes.
Antibiotics and Protein Synthesis
Differences between prokaryotic and eukaryotic translation provide targets for action of antibiotics. Puromycin, for example prevents prokaryotic protein synthesis by causing polypeptide changes to be released before translation is complete. The compound resembles the aminoacyl-tRNA molecule. Puromycin binds the A site and inhibits entry of aminoacyl-tRNA. The molecule's alpha amino group forms a peptide bond with the polypeptide in the P site, causing the polypeptide to be released prematurely. Another antibiotic, streptomycin, interferes with binding of formylmethionyl-tRNA to ribosomes, preventing initiation.
Another compound, diphtheria toxin produced by the bacterium Corynebacterium diphtheriae is lethal due to the ability of its A fragment to covalently modify EF2, effectively stopping translocation. The mechanism of this action is that the A fragment catalyzes transfer of adenosine diphosphate ribose from NAD+ to diphthalamide, a modified amino acid residue in EF2.