AUG Initiation Codon

Although AUG is the initiation codon normally used in bacteria, studies of binding of fMet-tRNAf to ribosomes (Clarck and Marcher, 1966) or translation of synthetic polynucleotides (Thach et ai, 1966) have revealed that GUG and, to a lesser extent, UUG are also functional initiation codons. A single case of initiation at an AUU codon is also known for the Escherichia coli initiation factor IF-3 protein (Sacerdot et ai, 1982). By contrast, a compilation of over 200 ribosome binding site sequences in eukaryotic mRNA (Kozak, 1981a, 1983) has yielded only AUG as initiation codon. It appears, therefore, that initiation of eukaryotic translation occurs exclusively at AUG codons. 

Usually, but not always, the functional AUG initiation codon is the first AUG codon encountered when reading the mRNA from its 5 end. This was first pointed out by Kozak (1978, 1981a) who could find, among 200 mRNA sequences, only 18 that contained AUG triplets upstream from the functional initiation site (Kozak, 1983). This led to the proposal that 40 S ribosomal subunits may scan the mRNA from its 5 end and simply initiate at the first AUG codon they encounter (Kozak, 1978). In poliovirus RNA, however, no fewer than seven AUG codons precede the 5 -proximal initiation site for translation (Kitamura et ai, 1981). To account for the exceptions, Kozak (1981 ) has proposed that sequences flanking the functional AUG codon may be important a purine, usually A, frequently occurs three residues before the AUG codon, while a purine, usually G, often follows the AUG sequence. Either of these features is lacking in some, but not all, of the upstream AUG codons in the exceptional mRNA species. 

As a rule, eukaryotic mRNA contains only a single functional initiation site for translation (Jacobson and Baltimore, 1968). This appears to be true for cellular mRNA and for many viral mRNA species. In the late 16 S mRNA of SV40, however, a 62-amino acid polypeptide, the agnoprotein, is encoded upstream from the capsid protein VPl (Jay et ai, 1981). Since this mRNA template expresses both proteins, late 16 S mRNA of SV40 is a true polycistronic mRNA containing two independent initiation sites. So far, it is the only unequivocal exception to the observation that eukaryotic mRNA is mono-cistronic, although evidence in support of the existence of two ini- 

This concept is supported by the findings that two functional, overlapping reading frames occur in influenza B virus RNA 6 (Shaw et al., 1983) and in an mRNA of human adenovirus (Bos et al., 1981). In both cases, two different polypeptide chains are encoded, starting at different AUG triplets. 

Finally, the issue of where initiation occurs in mRNA should be clearly separated from the question, how often initiation takes place. 

---

Figure 7.5 Model of ferritin (and erythroid a-aminolaevulinate synthase) translation/ribosome binding regulation by IRP. In (a), with IRP not bound to the IRE (1) binding of the 43S preinitiation complex (consisting of the small ribosomal 40S subunit, GTP and Met-tRNAMet) to the mRNA is assisted by initiation factors associated with this complex, as well as additional eukaryotic initiation factors (elFs) that interact with the mRNA to facilitate 43S association. Subsequently (2), the 43S preinitiation complex moves along the 5 -UTR towards the AUG initiator codon, (3) GTP is hydrolysed, initiation factors are released and assembly of the 80S ribosome occurs. Protein synthesis from the open reading frame (ORF) can now proceed. In (b) With IRP bound to the IRE, access of the 43S preinitiation complex to the mRNA is sterically blocked. From Gray and Hentze, 1994, by permission of Oxford University Press.

ALTERNATIVE START SITES If all of the above didn t provide enough diversity, some messages contain two AUG initiation codons separated by some intervening information. Protein synthesis can initiate at either site. This is useful for making proteins with or without NH2- terminal signal sequences. 

As opposed to the case in procaryotes, eucaryotic translation does not require a specific sequence for the binding of the ribosome. Procaryotes rely on the Shine-Dalgarno sequence, which is complementary to sequences of the 16S RNA of the 308 subunit. The Shine-Dalgamo sequence mediates the binding of mRNA to the 308 ribosome and ensures the correct positioning of the AUG initiation codon. 

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. 27-19). 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 methionine. 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 tRNAfMet. The amino acid incorporated in response to the (5 )AUG initiation codon is A7-formyl-methionine (fMet). It arrives at the ribosome as A7-formylmethionyl-tRNAfMet (fMet-tRNAfMet), which is formed in two successive reactions. First, methionine is attached to tRNAfMet by the Met-tRNA synthetase (which in E. coli aminoacylates both tRNAfMet and tRNAMet) ... 

A four-base overlap between dihydrofolate reductase and thymidylate synthase has been found201 in the DNA of phage T4. A transposable DNA insertion sequence (see Section D,5) in E. coli encodes two genes, one of which is contained within the other and which is transcribed from the opposite strand of DNA.202 The double-stranded RNA of a reovirus produces two peptides from the same sequence using two different AUG initiation codons in different reading frames 203... 

Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell, 44, 283-292. 

The first codon translated in all mRNAs is AUG which codes for methionine. This AUG is called the start codon or initiation codon. Naturally, other AUG codons also occur internally in an mRNA where they encode methionine residues internal to the protein. Two different tRNAs are used for these two types of AUG codon tRNAfMet is used for the initiation codon and is called the initiator tRNA whereas tRNAmMet is used for internal AUG codons. In prokaryotes the first amino acid of a new protein is /V-formylmethionine (abbreviated fMet). Hence the aminoacyl-tRNA used in initiation is fMet-tRNAfMet. It is essential that the correct AUG is used as the initiation codon since this sets the correct reading frame for translation (see Topic HI). A short sequence rich in purines (5 -AGGAGGU-3 ), called the Shine-Dalgarno sequence, lies 5 to the AUG initiation codon (Fig. 3) and is complementary to part of the 16S rRNA in the small ribo-somal subunit. Therefore this is the binding site for the 30S ribosomal subunit... 

The small subunit then binds to the mRNA via the Shine-Dalgarno sequence and moves 3 along the mRNA until it locates the AUG initiation codon. 

Eukaryotic ribosomes are larger (80S) and more complex than prokaryotic ribosomes (70S). Initiation is basically similar in prokaryotes and eukaryotes except that in eukaryotes at least nine initiation factors are involved (cf. three factors in prokaryotes), the initiating amino acid is methionine (cf. N-formylmethionine in prokaryotes), eukaryotic mRNAs do not contain Shine-Dalgarno sequences (so the AUG initiation codon is detected by the ribosome scanning instead), and eukaryotic mRNA is monocistronic (cf. some polycistronic mRNAs in prokaryotes). Initiation in eukaryotes involves the formation of a 48S preinitiation complex between the 40S ribosomal subunit, mRNA, initiation factors and Met-tRNA 61. The ribosome then scans the mRNA to locate the AUG initiation codon. The 60S ribosomal subunit now binds to form the 80S initation complex. 

Both prokaryotes and eukaryotes initiate protein synthesis with a specialized methionyl-tRNA in response to an AUG initiation codon. Eukaryotes, however, use an initiator met—tRNAmeti—that is not formylated. Recognition of the initiator AUG is also different. Only one coding sequence exists per eukaryotic mRNA, and eukaryotic mRNAs are capped. Initiation, therefore, uses a specialized capbinding initiation factor to position the mRNA on the small riboso-mal subunit. Usually, the first AUG after the cap (that is, 3 to it) is used for initiation. 

A list of key differences between prokaryotes and eukaryotes with respect to protein synthesis is shown in Table 9-1. These include the existence of multiple eukaryotic initiation factors that facilitate the assembly of the riboso-mal protein synthetic machinery, whereas there are only three for prokaryotes. An initiation site on bacterial mRNA consists of the AUG initiation codon preceded with a gap of approximately 10 bases by the Shine-Dalgamo polypurine hexamer, whereas the 5 Cap (a 7-methylguanylate residue in a 5 —>5 triphosphate linkage) acts as an initiation signal in eukaryotes. In prokaryotes, the first or A-terminal amino acid is a formyl-methionine (fMet), but in eukaryotes it is usually a simple methionine. Additionally, the size and nature of the prokaryotic ribosomes are quite different from the eukaryotic ribosomes. 

As shown in Figure 26.10, a purine-rich sequence, called the Shine-Dalgarno sequence, is centered 10 nucleotides upstream of the AUG codon in most bacterial mRNAs. It was discovered that the 30 end of bacterial 16S rRNA has a complementary sequence which can base pair with the Shine-Dalgarno sequence to position the 30S subunit relative to the initiator AUG codon. Ribosome positioning in eukaryotes is more difficult to predict, but there are two general rules for identifying the most likely eukaryotic AUG initiator codon (1) it is often the first AUG encountered by the ribosome after it binds the 50 mRNA cap and "scans" in the 30 direction and (2) there are preferred contexts for the initiator AUG which have been defined by the consensus sequence GCCPuCCAUGG. 

The SOS subunit binds to the 30S initiation complex. It contains three sites for tRNA binding, called the P site (peptidyl), the A site (aminoacyl), and the E site (exit). When the two ribosomal subunits join, the AUG initiator codon with its bound tRNAf t aligns with the P site. 

The double-stranded RNA of a reovirus produces two peptides from the same sequence using two different AUG initiation codons in different reading frames. ... 

FIGURE 12.11 Various Shine-Dalgamo sequences recognized by E. coli ribosomes. These sequences lie about 10 nucleotides upstream from their respective AUG initiation codon and are complementary to the UCCU core sequence element of E. coli 16S rRNA. G U as well as canonical G C and A U base pairs are involved here. 

These experiments have been dissapointing in the sense that the only features which seem to be universal are the AUG initiation codon and the 5 cap structure (7I, 72), and even the latter is missing from some viral RNAs, notably picornavirus RNAs (both the virion RNA (75) and. the viral mRNA (74)), Cowpea Mosaic Virus (CPMV) RNA (75)f and Satellite Tobacco Necrosis (STNV) RNA (76). The role of the cap has been studied by examining the properties of mRNA... 

How is the correct AUG initiation codon selected in the second stage of initiation, in moving from the cap to the initiation site ... 

The phasing of the ribosome to the AUG initiation codon - the final stage in initiation site selection - gives rise to fewer conceptual problems, because the initiating ribosome binds Met-tENA before associating with the mENA. As we pointed out when this was first discovered (16), this means that the anticodon of Met-tENA can assist in the phasing of the ribosome precisely to the initiation codon, through normal codon-anticodon interaction. 

After the ribosome has been assembled, with the initiator tRNA bound at the P site and occupying the AUG initiator codon, the next amino acyl tRNA binds to the A site of the ribosome, with its anticodon bound to the next codon in the sequence. 

Initiation A multistep reaction between ribosomal subunits, charged initiator transfer RNA, and messenger RNA that results in apposition of the ribosome-bound initiator Met-tRNA with an AUG initiator codon in mRNA. In this position, the ribosome is poised to form the first peptide bond. 

The 5 leader in eukaryotic mRNA is defined as the sequence between the cap and the AUG initiation codon. The most striking... 

---