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Electron micrographs of (A) 30S subunits, (B) 50S subunits, and (C) 70S ribosomes. [Courtesy of Dr. James Lake.]
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We turn now to ribosomes, the molecular machines that coordinate the interplay of charged tRNAs, mRNA, and proteins that leads to protein synthesis. An E. coli ribosome is a ribonucleoprotein assembly with a mass of about 2700 kd, a diameter of approximately 200 Å, and a sedimentation coefficient of 70S. The 20,000 ribosomes in a bacterial cell constitute nearly a fourth of its mass.
Electron micrographs of (A) 30S subunits, (B) 50S subunits, and (C) 70S ribosomes. [Courtesy of Dr. James Lake.]
). These subunits can be further split into their constituent proteins and RNAs. The 30S subunit contains 21 different proteins (referred to as S1 through S21) and a 16S RNA molecule. The 50S subunit contains 34 different proteins (L1 through L34) and two RNA molecules, a 23S and a 5S species. A ribosome contains one copy of each RNA molecule, two copies of the L7 and L12 proteins, and one copy of each of the other proteins. The L7 protein is identical with L12 except that its amino terminus is acetylated. Only one protein is common to both subunits: S20 is identical with L26. Both the 30S and the 50S subunits can be reconstituted in vitro from their constituent proteins and RNA, as was first achieved by Masayasu Nomura in 1968. This reconstitution is an outstanding example of the principle that supramolecular complexes can form spontaneously from their macromolecular constituents.
Detailed models of the ribosome based on the results of x-ray crystallographic studies of the 70S ribosome and the 30S and 50S subunits. 23S RNA is shown in yellow, 5S RNA in orange, 16S RNA in green, proteins of the 50S subunit in red, and proteins of the 30S subunit in blue.
). The determination of this structure requires the positioning of more than 100,000 atoms. The features of these structures are in remarkable agreement with interpretations of less-direct experimental probes. These structures provide an invaluable framework for examining the mechanism of protein synthesis.
(A) The secondary structure of 16S ribosomal RNA deduced from sequence comparison and the results of chemical studies. (B) The tertiary structure of 16S RNA determined by x-ray crystallography. [Part A courtesy of Dr. Bryn Weiser and Dr. Harry Noller.]
). The striking finding is that ribosomal RNAs (rRNAs) are folded into defined structures with many short duplex regions. This conclusion and essentially all features of the secondary structure have been confirmed by the x-ray crystallographically determined structures.
The structure of ribosomal protein L19 of the 50S ribosomal subunit reveals a long segment of extended structure that fits through some of the cavities within the 23S RNA molecule.
For many years, ribosomal proteins were presumed to orchestrate protein synthesis and ribosomal RNAs were presumed to serve primarily as structural scaffolding. The current view is almost the reverse. The discovery of catalytic RNA made biochemists receptive to the possibility that RNA plays a much more active role in ribosomal function. The detailed structures make it clear that the key sites in the ribosome are composed almost entirely of RNA. Contributions from the proteins are minor. Many of the proteins have elongated structures that “snake” their way into the RNA matrix (Figure 29.18). The almost inescapable conclusion is that the ribosome initially consisted only of RNA and that the proteins were added later to fine tune its functional properties. This conclusion has the pleasing consequence of dodging a “chicken and egg” question—namely, How can complex proteins be synthesized if complex proteins are required for protein synthesis?
Before the mechanism of protein synthesis could be examined, several key facts had to be established. The results of pulse-labeling studies by Howard Dintzis established that protein synthesis proceeds sequentially from the amino terminus. Reticulocytes (young red blood cells) that were actively synthesizing hemoglobin were treated with [3H] leucine. In a period of time shorter than that required to synthesize a complete chain, samples of hemoglobin were taken, separated into α and β chains, and analyzed for the distribution of 3H within their sequences. In the earliest samples, only regions near the carboxyl ends contained radioactivity. In later samples, radioactivity was present closer to the amino terminus as well. This distribution is the one expected if the amino-terminal regions of some chains had already been partly synthesized before the addition of the radioactive amino acid. Thus, protein synthesis begins at the amino terminus and extends toward the carboxyl terminus.
The sequence of amino acids in a protein is translated from the nucleotide sequence in mRNA. In which direction is the message read? The answer was established by using the synthetic polynucleotide
as the template in a cell-free protein-synthesizing system. AAA encodes lysine, whereas AAC encodes asparagine. The polypeptide product was
Because asparagine was the carboxyl-terminal residue, we can conclude that the codon AAC was the last to be read. Hence, the direction of translation is 5′ → 3′.
Transcription of a segment of DNA from E. coli generates mRNA molecules that are immediately translated by multiple ribosomes. [From O. L. Miller, Jr., B. A. Hamkalo, and C. A. Thomas, Jr. Science 169(1970):392.]
).
How does protein synthesis start? The simplest possibility would be for the first three nucleotides of each mRNA to serve as the first codon; no special start signal would then be needed. However, the experimental fact is that translation does not begin immediately at the 5′ terminus of mRNA. Indeed, the first translated codon is nearly always more than 25 nucleotides away from the 5′ end. Furthermore, in prokaryotes, many mRNA molecules are polycistronic, or polygenic—that is, they encode two or more polypeptide chains. For example, a single mRNA molecule about 7000 nucleotides long specifies five enzymes in the biosynthetic pathway for tryptophan in E. coli. Each of these five proteins has its own start and stop signals on the mRNA. In fact, all known mRNA molecules contain signals that define the beginning and end of each encoded polypeptide chain.
Sequences of mRNA initiation sites for protein synthesis in some bacterial and viral mRNA molecules. Comparison of these sequences reveals some recurring features.
). In addition, each initiator region contains a purine-rich sequence centered about 10 nucleotides on the 5′ side of the initiator codon.
The role of this purine-rich region, called the Shine-Dalgarno sequence, became evident when the sequence of 16S rRNA was elucidated. The 3′ end of this rRNA component of the 30S subunit contains a sequence of several bases that is complementary to the purine-rich region in the initiator sites of mRNA. Mutagenesis of the CCUCC sequence near the 3′ end of 16S rRNA to ACACA markedly interferes with the recognition of start sites in mRNA. This and other evidence shows that the initiator region of mRNA binds to the 16S rRNA very near its 3′ end. The number of base pairs linking mRNA and 16S rRNA ranges from three to nine. Thus, two kinds of interactions determine where protein synthesis starts: (1) the pairing of mRNA bases with the 3′ end of 16S rRNA and (2) the pairing of the initiator codon on mRNA with the anticodon of an initiator tRNA molecule.
The methionine residue found at the amino-terminal end of E. coli proteins is usually modified. In fact, protein synthesis in bacteria starts with N-formylmethionine (fMet). A special tRNA brings formylmethionine to the ribosome to initiate protein synthesis. This initiator tRNA (abbreviated as tRNAf) differs from the one that inserts methionine in internal positions (abbreviated as tRNAm). The subscript “f” indicates that methionine attached to the initiator tRNA can be formylated, whereas it cannot be formyl-ated when attached to tRNAm. In approximately one-half of E. coli proteins, N-formylmethionine is removed when the nascent chain is 10 amino acids long.
Initiator tRNA (tRNAf) is first charged with methionine, and then a formyl group is transferred to the methionyl- tRNAf from N10-formyltetrahydrofolate.
). The activated formyl donor in this reaction is N10-formyltetrahydrofolate (Section 24.2.6). It is significant that free methionine and methionyl-tRNAm are not substrates for this transformylase.
(A) Three tRNA-binding sites are present on the 70S ribosome. They are called the A (for aminoacyl), P (for peptidyl), and E (for exit) sites. Each tRNA molecule contacts both the 30S and the 50S subunit. (B) The tRNA molecules in sites A and P are base paired with mRNA.
). As expected, the mRNA fragment is bound within the 30S subunit. Each of the tRNA molecules bridges between the 30S and 50S subunits. At the 30S end, two of the three tRNA molecules are bound to the mRNA fragment through anticodon-codon base pairs. These binding sites are called the A site (for aminoacyl) and the P site (for peptidyl). The third tRNA molecule is bound to an adjacent site called the E site (for exit).
A tunnel passes through the 50S subunit beginning at the site of peptide-bond formation (shown in blue). The growing polypeptide chain passes through this tunnel.
). The polypeptide chain passes through this tunnel during synthesis.
The cycle begins with peptidyl-tRNA in the P site. An aminoacyl-tRNA binds in the A site. With both sites occupied, a new peptide bond is formed. The tRNAs and the mRNA are translocated through the action of elongation factor G, which moves the deacylated tRNA to the E site. Once there, it is free to dissociate to complete the cycle.
)? The three sites in our snapshot of protein synthesis provide a clue. The initiator tRNA is bound in the P site on the ribosome. A charged tRNA with an anticodon complementary to the codon in the A site then binds. The stage is set for the formation of a peptide bond: the formylmethionine molecule linked to the initiator tRNA will be transferred to the amino group of the amino acid in the A site. The transfer takes place in a ribosome site called the peptidyl transferase center.
The amino group of the aminoacyl-tRNA attacks the carbonyl group of the ester linkage of the peptidyl-tRNA to form a tetrahedral intermediate. This intermediate collapses to form the peptide bond and release the deacylated tRNA.
). The peptidyl transferase center includes bases that promote this reaction by helping to form an -NH2 group on the A site aminoacyl-tRNA and by helping to stabilize the tetrahedral intermediate that forms. This reaction is, in many ways, analogous to the reverse of the reaction catalyzed by serine proteases such as chymotrypsin (Section 9.1.2). The peptidyl-tRNA is analogous to the acyl-enzyme form of a serine protease. In a serine protease, the acyl-enzyme is generated with the use of the free energy associated with cleaving an amide bond. In the ribosome, the free energy necessary to form the analogous species, an amino-acyl-tRNA, comes from the ATP that is cleaved by the aminoacyl-tRNA synthetase before the arrival of the tRNA at the ribosome.
With the peptide bond formed, the peptide chain is now attached to the tRNA in the A site on the 30S subunit while a change in the interaction with the 50S subunit has placed that tRNA and its peptide in the P site of the large subunit. The tRNA in the P site of the 30S subunit is now uncharged. For translation to proceed, the mRNA must be moved (or translo-cated) so that the codon for the next amino acid to be added is in the A site. This translocation takes place through the action of a protein enzyme called elongation factor G (Section 29.4.3), driven by the hydrolysis of GTP. On completion of this step, the peptidyl-tRNA is now fully in the P site, and the uncharged initiator tRNA is in the E site and has been disengaged from the mRNA. On dissociation of the initiator tRNA, the ribosome has returned to its initial state except that the peptide chain is attached to a different tRNA, the one corresponding to the first codon past the initiating AUG. Note that the peptide chain remains in the P site on the 50S subunit throughout this cycle, presumably growing into the tunnel. This cycle is repeated as new aminoacyl-tRNAs move into the A site, allowing the polypeptide to be elongated indefinitely.
With a free terminal amino group, dipeptidyl-tRNA can cyclize to cleave itself from tRNA. Formylation of the amino terminus blocks this reaction.
). Suppose that the amino-terminus is not blocked. After the first peptidyl-transfer reaction, a dipeptide is linked to the tRNA in the P site. If a free amino group is present in the terminal amino acid, this amino group can attack the carbonyl group of the ester linkage to the tRNA, forming a very stable six-membered ring and terminating translation.
On the basis of the mechanism described in Section 29.3.7, the base-pairing interaction between the anticodon on the incoming tRNA and the codon in the A site on mRNA determines which amino acid is added to the polypeptide chain. Does the amino acid attached to the tRNA play any role in this process? This question was answered in the following way. First, cysteine was attached to its cognate tRNA. The attached cysteine unit was then converted into alanine by adding Raney nickel to Cys-tRNACys; the reaction removed the sulfur atom from the cysteine residue without affecting its linkage to tRNA. Thus, a mischarged aminoacyl-tRNA was produced in which alanine was covalently attached to a tRNA specific for cysteine.
Does this mischarged tRNA recognize the codon for cysteine or for alanine? The answer came when the tRNA was added to a cell-free protein-synthesizing system. The template was a random copolymer of U and G in the ratio of 5:1, which normally incorporates cysteine (encoded by UGU) but not alanine (encoded by GCN). However, alanine was incorporated into a polypeptide when Ala-tRNACys was added to the incubation mixture. The same result was obtained when mRNA for hemoglobin served as the template and [14C]alanyl-tRNACys was used as the mischarged aminoacyl-tRNA. The only radioactive tryptic peptide produced was one that normally contained cysteine but not alanine. Thus, the amino acid in aminoacyl-tRNA does not play a role in selecting a codon.
In recent years, the ability of mischarged tRNAs to transfer their amino acid cargo to a growing polypeptide chain has been used to synthesize peptides with amino acids not found in proteins incorporated into specific sites in a protein. Aminoacyl-tRNAs are first linked to these unnatural amino acids by chemical methods. These mischarged aminoacyl-tRNAs are added to a cell-free protein-synthesizing system along with specially engineered mRNA that contains codons corresponding to the anticodons of the mischarged aminoacyl-tRNAs in the desired positions. The proteins produced have unnatural amino acids in the expected positions. More than 100 different unnatural amino acids have been incorporated in this way. However, only l-amino acids can be used; apparently this stereochemistry is required for peptide-bond formation to take place.
What are the rules that govern the recognition of a codon by the anticodon of a tRNA? A simple hypothesis is that each of the bases of the codon forms a Watson-Crick type of base pair with a complementary base on the anticodon. The codon and anticodon would then be lined up in an antiparallel fashion. In the diagram in the margin, the prime denotes the complementary base. Thus X and X′ would be either A and U (or U and A) or G and C (or C and G). According to this model, a particular anticodon can recognize only one codon.
| First base of anticodon | Third base of codon |
|---|---|
| C | G |
| A | U |
| U | A or G |
| G | U or C |
| I | U, C, or A |
The wobble hypothesis is now firmly established. The anticodons of tRNAs of known sequence bind to the codons predicted by this hypothesis. For example, the anticodon of yeast alanyl-tRNA is IGC. This tRNA recognizes the codons GCU, GCC, and GCA. Recall that, by convention, nucleotide sequences are written in the 5′ → 3′ direction unless otherwise noted. Hence, I (the 5′ base of this anticodon) pairs with U, C, or A (the 3′ base of the codon), as predicted.
Two generalizations concerning the codon-anticodon interaction can be made:
The first two bases of a codon pair in the standard way. Recognition is precise. Hence, codons that differ in either of their first two bases must be recognized by different tRNAs. For example, both UUA and CUA encode leucine but are read by different tRNAs.
The first base of an anticodon determines whether a particular tRNA molecule reads one, two, or three kinds of codons: C or A (one codon), U or G (two codons), or I (three codons). Thus, part of the degeneracy of the genetic code arises from imprecision (wobble) in the pairing of the third base of the codon with the first base of the anticodon. We see here a strong reason for the frequent appearance of inosine, one of the unusual nucleosides, in anticodons. Inosine maximizes the number of codons that can be read by a particular tRNA molecule. The inosines in tRNA are formed by deamination of adenosine after synthesis of the primary transcript.
Why is wobble tolerated in the third position of the codon but not in the first two? The 30S subunit has two adenine bases (A1492 and A1493 in the 16S RNA) that form hydrogen bonds on the minor-groove side of the codon-anticodon duplex. These interactions serve to check whether Watson-Crick base pairs are present in the first two positions of the codon- anticodon duplex. No such inspection device is present for the third position so more-varied base pairs are tolerated. This mechanism for ensuring fidelity is analogous to the minor-groove interactions utilized by DNA polymerase for a similar purpose (Section 27.2.3). Thus, the ribosome plays an active role in decoding the codon-anticodon interactions.
