Protein Synthesis: A Specific Amino Acid Initiates Protein Synthesis
Protein synthesis begins at the amino-terminal end and proceeds by the stepwise addition of amino acids to the carboxyl-terminal end of the growing polypeptide, as determined by Howard Dintzis in 1961 (Fig. 2). The AUG initiation codon thus specifies an amino-terminal methionine residue. Although methionine has only one codon, (5) AUG, all organisms have two tRNAs for me thionine. One is used exclusively when (5) AUG is the initiation codon for protein synthesis. The other is used to code for a Met residue in an internal position in a polypeptide. The distinction between an initiating (5) AUG and an internal one is straightforward. In bacteria, the two types of tRNA specific for methionine are designated tRNAMet and tRNAMet. The amino acid incorporated in response to the (5) AUG initiation codon is N-formyl methionine (fMet). It arrives at the ribosome as N-formyl methionyl-tRNAfMet (fMet-tRNAMet), which is formed in two successive reactions. First, methionine is attached to tRNAMet by the Met-tRNA synthetase (which in E. coli aminoacylates both tRNAMet and tRNAMet):
Methionine+ tRNAfMet +ATP→ Met-tRNAfMet + AMP +PPi
Next, a transformylase transfers a formyl group from N10-formyltetrahydrofolate to the amino group of the Met residue:
N10-Formyltetrahydrofolate+Met-tRNAfMet → tetrahydrofolate+ fMet- tRNAfMet
The transformylase is more selective than the Met-tRNA synthetase; it is specific for Met residues attached to tRNAfMet, presumably recognizing some unique structural feature of that tRNA. By contrast, Met-tRNAMet inserts methionine in interior positions in polypeptides. Addition of the N-formyl group to the amino group of methionine by the transformylase prevents fMet from entering interior positions in a polypeptide while also allowing fMet-tRNAfMet to be bound at a specific ribosomal initiation site that accepts neither Met-tRNAMet nor any other aminoacyl-tRNA.
In eukaryotic cells, all polypeptides synthesized by cytosolic ribosomes begin with a Met residue (rather than fMet), but, again, the cell uses a specialized initiating
tRNA that is distinct from the tRNAMet used at (5) AUG codons at interior positions in the mRNA. Polypeptides synthesized by mitochondrial and chloroplast ribosomes, however, begin with N-formylmethionine. This strongly supports the view that mitochondria and chloroplasts originated from bacterial ancestors that were symbiotically incorporated into precursor eukaryotic cells at an early stage of evolution . How can the single (5) AUG codon distinguish between the starting N-formylmethionine (or methionine, in eukaryotes) and interior Met residues? The details of the initiation process provide the answer.
The Three Steps of Initiation The initiation of polypep tide synthesis in bacteria requires (1) the 30S ribosomal subunit, (2) the mRNA coding for the polypeptide to be made, (3) the initiating fMet-tRNAfMet, (4) a set of three proteins called initiation factors (IF-1, IF-2, and IF-3), (5) GTP, (6) the 50S ribosomal subunit, and (7) Mg2+. Formation of the initiation complex takes place in three steps (Fig. 3). In step 1 the 30S ribosomal subunit binds two initiation factors, IF-1 and IF-3. Factor IF-3 prevents the 30S and 50S subunits from combining prematurely. The mRNA then binds to the 30S subunit. The initiating (5) AUG is guided to its correct position by the Shine Dalgarno sequence (named for Australian researchers John Shine and Lynn Dalgarno, who identified it) in the mRNA. This consensus sequence is an initiation signal of four to nine purine residues, 8 to 13 bp to the 5 side of the initiation codon (Fig. 4a). The sequence base-pairs with a complementary pyrimidine-rich sequence near the 3end of the 16S rRNA of the 30S ribosomal subunit (Fig. 4b). This mRNA-rRNA in teraction positions the initiating (5) AUG sequence of the mRNA in the precise position on the 30S subunit where it is required for initiation of translation. The particular (5) AUG where fMet-tRNAfMet is to be bound is distinguished from other methionine codons by its proximity to the Shine-Dalgarno sequence in the mRNA. Bacterial ribosomes have three sites that bind aminoacyl-tRNAs, the aminoacyl (A) site, the peptidyl (P) site, and the exit (E) site. Both the 30S and the 50S subunits contribute to the characteristics of the A and P sites, whereas the E site is largely con fined to the 50S subunit. The initiating (5) AUG is positioned at the P site, the only site to which fMet tRNAfMet can bind (Fig. 3). The fMet-tRNAfMet is the only aminoacyl-tRNA that binds first to the P site; during the subsequent elongation stage, all other in coming aminoacyl-tRNAs (including the Met-tRNAMet that binds to interior AUG codons) bind first to the A site and only subsequently to the P and E sites. The E site is the site from which the “uncharged” tRNAs leave during elongation. Factor IF-1 binds at the A site and prevents tRNA binding at this site during initiation.
In step 2 of the initiation process (Fig. 3), the complex consisting of the 30S ribosomal subunit, IF-3, and mRNA is joined by both GTP-bound IF-2 and the initiating fMet-tRNAfMet. The anticodon of this tRNA now pairs correctly with the mRNA’s initiation codon. In step 3 this large complex combines with the 50S ribosomal subunit; simultaneously, the GTP bound to IF-2 is hydrolyzed to GDP and Pi, which are released from the complex. All three initiation factors depart from the ribosome at this point. Completion of the steps in Figure 3 produces a functional 70S ribosome called the initiation com plex, containing the mRNA and the initiating fMet tRNAfMet. The correct binding of the fMet-tRNAfMet to the P site in the complete 70S initiation complex is assured by at least three points of recognition and attachment: the codon-anticodon interaction involving the initiation AUG fixed in the P site; interaction between the Shine-Dalgarno sequence in the mRNA and the 16S rRNA; and binding interactions between the ribosomal P site and the fMet-tRNAfMet. The initiation complex is now ready for elongation.
Initiation in Eukaryotic Cells Translation is generally similar in eukaryotic and bacterial cells; most of the significant differences are in the mechanism of initiation. Eu karyotic mRNAs are bound to the ribosome as a complex with a number of specific binding proteins. Several of these tie together the 5 and 3 ends of the message. At the 3end, the mRNA is bound by the poly(A) binding protein (PAB). Eukaryotic cells have at least nine initiation factors. A complex called eIF4F, which includes the proteins eIF4E, eIF4G, and eIF4A, binds to the 5cap through eIF4E. The protein eIF4G binds to both eIF4E and PAB, effectively tying them to gether (Fig. 5). The protein eIF4A has an RNA helicase activity. It is the eIF4F complex that associates with another factor, eIF3, and with the 40S ribosomal subunit. The efficiency of translation is affected by many properties of the mRNA and proteins in this complex, including the length of the 3 poly(A) tract (in most cases, longer is better). The end-to-end arrangement of the eukaryotic mRNA facilitates translational regulation of gene expression, considered in Chapter 28. The initiating (5) AUG is detected within the mRNA not by its proximity to a Shine-Dalgarno-like sequence but by a scanning process: a scan of the mRNA from the 5end until the first AUG is encountered, signaling the beginning of the reading frame. The eIF4F complex is probably involved in this process, perhaps using the RNA helicase activity of eIF4A to eliminate secondary structure in the 5 untranslated portion of the mRNA. Scanning is also facilitated by another protein, eIF4B. The roles of the various bacterial and eukaryotic initiation factors in the overall process are summarized in Table 27–8. The mechanism by which these proteins act is an important area of investigation.
FIGURE 1 Structural elements of tRNAAla that are required for recognition by Ala-tRNA synthetase. (a) The tRNAAla structural elements recognized by the Ala-tRNA synthetase are unusually simple. A single G=U base pair (pink) is the only element needed for specific binding and aminoacylation. (b) A short synthetic RNA minihelix, with the critical G=U base pair but lacking most of the remaining tRNA structure. This is specifically aminoacylated with alanine almost as efficiently as the complete tRNAAla.
FIGURE 2 Proof that polypeptides grow by addition of amino acid residues to the carboxyl end: the Dintzis experiment. Reticulocytes (immature erythrocytes) actively synthesizing hemoglobin were incubated with radioactive leucine (selected because it occurs frequently in both the - and -globin chains). Samples of completed chains were isolated from the reticulocytes at various times afterward, and the distribution of radioactivity was determined. The dark red zones show the portions of completed -globin chains containing radioactive Leu residues. At 4 min, only a few residues at the carboxyl end of -globin were labeled, because the only complete globin chains with incorporated label after 4 min were those that had nearly completed synthesis at the time the label was added. With longer incubation times, successively longer segments of the polypeptide contained labeled residues, always in a block at the carboxyl end of the chain. The unlabeled end of the polypeptide (the amino terminus) was thus defined as the initiating end, which means that polypeptides grow by successive addition of amino acids to the carboxyl end.
FIGURE 3 Formation of the initiation complex in bacteria. The complex forms in three steps (described in the text) at the expense of the hydrolysis of GTP to GDP and Pi. IF-1, IF-2, and IF-3 are initiation factors. P designates the peptidyl site, A the aminoacyl site, and E the exit site. Here the anticodon of the tRNA is oriented 3 to 5, left to right, as in Figure 27–8 but opposite to the orientation in Figures 27–16 and 27–18.
FIGURE 4 Messenger RNA sequences that serve as signals for initiation of protein synthesis in bacteria. (a) Alignment of the initiating AUG (shaded in green) at its correct location on the 30S ribosomal subunit depends in part on upstream Shine-Dalgarno sequences (pink). Portions of the mRNA transcripts of five prokaryotic genes are shown. Note the unusual example of the E. coliLacI protein, which initiates with a GUG (Val) codon (see Box 27–2). (b) The Shine Dalgarno sequence of the mRNA pairs with a sequence near the 3end of the 16S rRNA.
FIGURE 5 Protein complexes in the formation of a eukaryotic initiation complex. The 3 and 5 ends of eukaryotic mRNAs are linked by a complex of proteins that includes several initiation factors and the poly(A) binding protein (PAB). The factors eIF4E and eIF4G are part of a larger complex called eIF4F. This complex binds to the 40S ribosomal subunit.
