Analysis of ribosome binding sites from the s1 message of reovirus: Initiation at the first and second AUG codons

Two ribosome-protected initiation sites from the s1 message of reovirus have been characterized. Comparison of these sites with the previously determined sequence of s1 mRNA (Li et al., 1980) reveals that wheat germ ribosomes select and protect the first two AUG triplets in that message. This is unusual, since ribosomes initiate at a single site, the 5′-proximal AUG, in almost all other eukaryotic messenger RNAs that have been examined. The first AUG codon in s1 mRNA is preceded by a pyrimidine in position ?3, thus distinguishing it from most other eukaryotic messages, which have a purine (usually A) in that position. The behavior of s1 mRNA is consistent with the hypothesis that flanking nucleotides modulate the efficiency with which the migrating 40 S ribosomal subunit recognizes an AUG codon as a stop signal. If the first AUG triplet is flanked by suboptimal sequences, as in s1 mRNA, some 40 S ribosomes bypass that site and initiate at the next AUG downstream. The second AUG in the s1 message conforms to the consensus sequence (A-N-N-A-U-G-G) for eukaryotic initiation sites.     

Identification of features in 5' terminal fragments from reovirus mRNA which are important for ribosome binding

Four types of experiments were carried out with reovirus messenger RNAs or with 5′ terminal fragments of known sequence to identify features in mRNA which appear to be important for formation of initiation complexes with ribosomes. With a number of reovirus mRNAs, 40S initiation complexes had been previously shown to protect a significantly larger segment of the RNA (including the 5′ terminal m7G) than that protected by 80S initiation complexes. Each 80S-protected sequence had an AUG codon and was a subset of the 40S-protected sequence from the same message. When 40S- and 80S-protected fragments were tested for ability to rebind to ribosomes, the 80S-protected fragments showed considerably lower binding ability, implying that the “extra” sequences protected by 40S initiation complexes contribute to ribosome attachment. Nevertheless, wheat germ ribosomes select the same 5′ terminal initiation site in each reovirus mRNA, irrespective of the presence or absence of m7G on the message. This was demonstrated by comparing fingerprints of the ribosome-protected regions obtained with methylated versus unmethylated RNA. The contribution of m7G to formation of initiation complexes is therefore quantitative rather than qualitative. Limited T1 RNAase digestion of isolated 5′ terminal fragments from several reovirus messages generated a series of smaller fragments which were analyzed for ability to rebind to ribosomes. Partial digestion products up to 30 nucleotides in length which retained the 5′ cap but not the AUG codon were unable to associate stably with ribosomes, whereas every AUG-containing fragment that was analyzed was able to form initiation complexes. The efficiency of binding of certain AUG-containing fragments, however, was reduced by removal of either the 5′ terminal region, including the cap, or of sequences comprising the beginning of the coding region, on the 3′ side of the AUG. Complex formation between messenger RNA and ribosomes was inhibited by the trinucleotide AUG, but not by various other oligonucleotides. Although the inhibition was specific, a vast excess of trinucleotide was required for moderate inhibition of 80S complex formation, and the same concentration of AUG failed to inhibit formation of 40S initiation complexes.     

eIF5 and eIF5B together stimulate 48S initiation complex formation during ribosomal scanning

48S initiation complex (48S IC) formation is the first stage in the eukaryotic translation process. According to the canonical mechanism, 40S ribosomal subunit binds to the 5′-end of messenger RNA (mRNA) and scans its 5′-untranslated region (5′-UTR) to the initiation codon where it forms the 48S IC. Entire process is mediated by initiation factors. Here we show that eIF5 and eIF5B together stimulate 48S IC formation influencing initiation codon selection during ribosomal scanning. Initiation on non-optimal start codons—following structured 5′-UTRs, in bad AUG context, within few nucleotides from 5′-end of mRNA and CUG start codon—is the most affected. eIF5-induced hydrolysis of eIF2-bound GTP is essential for stimulation. GTP hydrolysis increases the probability that scanning ribosomal complexes will recognize and arrest scanning at a non-optimal initiation codon. Such 48S ICs are less stable owing to dissociation of eIF2*GDP from initiator tRNA, and eIF5B is then required to stabilize the initiator tRNA in the P site of 40S subunit. Alternative model that eIF5 and eIF5B cause 43S pre-initiation complex rearrangement favoring more efficient initiation codon recognition during ribosomal scanning is equally possible. Mutational analysis of eIF1A and eIF5B revealed distinct functions of eIF5B in 48S IC formation and subunit joining.     

Ribosomes bind leaderless mRNA in Escherichia coli through recognition of their 5'-terminal AUG

Leaderless mRNAs are translated in the absence of upstream signals that normally contribute to ribosome binding and translation efficiency. In order to identify ribosomal components that interact with leaderless mRNA, a fragment of leaderless cI mRNA from bacteriophage λ, with a 4-thiouridine (4^S-U) substituted at the +2 position of the AUG start codon, was used to form cross-links to Escherichia coli ribosomes during binary (mRNA+ribosome) and ternary (mRNA+ribosome+initiator tRNA) complex formation. Ribosome binding assays (i.e., toeprints) demonstrated tRNA-dependent binding of leaderless mRNA to ribosomes; however, cross-links between the start codon and 30S subunit rRNA and r-proteins formed independent of initiator tRNA. Toeprints revealed that a leaderless mRNA's 5′-AUG is required for stable binding. Furthermore, the addition of a 5′-terminal AUG triplet to a random RNA fragment can make it both competent and competitive for ribosome binding, suggesting that a leaderless mRNA's start codon is a major feature for ribosome interaction. Cross-linking assays indicate that a subset of 30S subunit r-proteins, located at either end of the mRNA tunnel, contribute to tRNA-independent contacts and/or interactions with a leaderless mRNA's start codon. The interaction of leaderless mRNA with ribosomes may reveal features of mRNA binding and AUG recognition that are distinct from known signals but are important for translation initiation of all mRNAs.     

Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes.

Sequences flanking the initiator codon in eukaryotic mRNAs are not random. Out of 153 messages examined, 151 have either a purine in position -3, or a G in position +4, or both. Thus, [A/G]XXAUGG emerges as the favored sequence for eukaryotic initiation sites. Nucleotides flanking nonfunctional AUG triplets, which occur in the 5'-noncoding region of a few eukaryotic messages, are different from those found at most functional sites. Whereas most authentic initiator codons are preceded by a purine (usually A) in position -3, most nonfunctional AUGs have a pyrimidine in that position. The observed asymmetry suggests that purines in positions -3 and +4 might facilitate recognition of the AUG condon during formation of initiation complexes. To test this idea, in vitro binding studies were carried out with 32P-labeled oligonucleotides. Binding of AUG-containing oligonucleotides to wheat germ ribosomes was significantly enhanced by placing a purine in position -3 or +4. The scanning model, which postulates that 40S ribosomal subunits attach at the 5'-end of a message and migrate down to the AUG codon, is discussed in light of these new observations. A modified version of the scanning mechanism is proposed.     

Secondary structure of mouse and rabbit α- and β-globin mRNAs: Differential accessibility of α and β initiator AUG codons towards nucleases

The nucleotide sequence from the 5′ terminus inward of one third of mouse α- and β^maj-globin messenger RNAs has been established. In addition, using 5′ ³²P end-labeled mRNAs as substrates and S1 and T1 nucleases as probes for single-stranded regions, the secondary structures of mouse and rabbit α- and β-globin mRNAs have been analyzed. Our results indicate that the AUG initiator codon in both mouse and rabbit β-globin mRNA is quite susceptible to cleavage with S1 and T1 nucleases, suggesting that it resides in a single-stranded exposed region. In contrast, the initiator AUG in the α-globin mRNA of both species is inaccessible to cleavage, indicating that it is either buried by tertiary structure or is base-paired. Since the rate of initiation of protein synthesis with β-globin mRNA in rabbit reticulocyte is 30–40% faster than for α-globin mRNA, these results imply a possible correlation between the differential rates of initiation with these two mRNAs and the accessibility of the respective AUG initiator codons.     

Genetic selection for mutations that reduce or abolish ribosomal recognition of the HIS4 translational initiator region.

A unique genetic selection was devised at the HIS4 locus to address the mechanism of translation initiation in Saccharomyces cerevisiae and to probe sequence requirements at the normal translational initiator region that might participate in ribosomal recognition of the AUG start codon. The first AUG codon at the 5' end of the HIS4 message serves as the start site for translation, and the -3 and +4 nucleotide positions flanking this AUG (AXXAUGG) correspond to a eucaryotic consensus start region. Despite this similarity, direct selection for mutations that reduce or abolish ribosomal recognition of this region does not provide any insight into the functional nature of flanking nucleotides. The only mutations identified that affected recognition of this region were alterations in the AUG start codon. Among 150 spontaneous isolates, 26 were shown to contain mutations in the AUG start codon, including all +1 changes (CUG, GUG, and UUG), all +3 changes (AUA, AUC, and AUU), and one +2 change (ACG). These seven mutations of the AUG start codon, as well as AAG and AGG constructed in vitro, were assayed for their ability to support HIS4 expression. No codon other than AUG is physiologically relevant to translation initiation at HIS4 as determined by growth tests and quantitated in his4-lacZ fusion strains. These data and analysis of other his4 alleles are consistent with a mechanism of initiation at HIS4 as proposed in the scanning model whereby the first AUG codon nearest the 5' end of the message serves as the start site for translation and points to the AUG codon in S. cerevisiae as an important component for ribosomal recognition of the initiator region.     

Interaction of bovine mitochondrial ribosomes with messenger RNA

The gene for subunit II of cytochrome oxidase (CoII) from bovine mitochondria has been cloned behind a T7 promoter and the corresponding mRNA synthesized in vitro. The RNA transcribed from this vector has a single nucleotide 5' to the start AUG and, thus, corresponds closely to the native mRNA. It binds to the small 28 S ribosomal subunit of bovine mitochondria but not to the large (39 S) subunit or to 55 S ribosomes. The binding occurs readily in the absence of auxiliary initiation factors or initiator tRNA. The complex formed appears to contain 1 mRNA/28 S subunit. The observed binding is specific for mRNA since neither tRNA nor ribosomal RNA can act as competitive inhibitors. The interaction of the mRNA with the 28 S subunit does not require an AUG codon near the 5' end and constructs containing 5' leaders of more than 100 nucleotides still bind efficiently. About 5% of the bound mRNA is protected from digestion by T1 RNase. The protected fragments do not arise from a specific region of the mRNA since they hybridize to several restriction fragments of the cloned CoII gene.     

The scanning model for translation: an update

The small (40S) subunit of eukaryotic ribosomes is believed to bind initially at the capped 5'-end of messenger RNA and then migrate, stopping at the first AUG codon in a favorable context for initiating translation. The first-AUG rule is not absolute, but there are rules for breaking the rule. Some anomalous observations that seemed to contradict the scanning mechanism now appear to be artifacts. A few genuine anomalies remain unexplained.     

Sliding of a 43S ribosomal complex from the recognized AUG codon triggered by a delay in eIF2-bound GTP hydrolysis

During eukaryotic translation initiation, 43S ribosomal complex scans mRNA leader unless an AUG codon in an appropriate context is found. Establishing the stable codon–anticodon base-pairing traps the ribosome on the initiator codon and triggers structural rearrangements, which lead to P_i release from the eIF2-bound GTP. It is generally accepted that AUG recognition by the scanning 43S complex sets the final point in the process of start codon selection, while latter stages do not contribute to this process. Here we use translation reconstitution approach and kinetic toe-printing assay to show that after the 48S complex is formed on an AUG codon, in case GTP hydrolysis is impaired, the ribosomal subunit is capable to resume scanning and slides downstream to the next AUG. In contrast to leaky scanning, this sliding is not limited to AUGs in poor nucleotide contexts and occurs after a relatively long pause at the recognized AUG. Thus, recognition of an AUG per se does not inevitably lead to this codon being selected for initiation of protein synthesis. Instead, it is eIF5-induced GTP hydrolysis and P_i release that irreversibly trap the 48S complex, and this complex is further stabilized by eIF5B and 60S joining.     

Protein synthesis in yeast Saccharomyces cerevisiae. Purification of Co-eIF-2A and 'mRNA-binding factor(s)' and studies of their roles in Met-tRNAf.40S.mRNA complex formation

Antibodies prepared against a homogeneous preparation of Co-eIF-2A20 [Ahmad et al. (1985) J. Biol. Chem. 260, 6955-6959] reacted with several polypeptides including an 80-kDa polypeptide present in a crude yeast ribosomal salt wash. This 80-kDa polypeptide, containing Co-eIF-2A (Co-eIF-2A80) activity, has been extensively purified using a two-step purification procedure involving an immunoaffinity column chromatograph prepared using antibodies against Co-eIF-2A20 (fraction II) and hydroxyapatite chromatography (fraction III). The factors, eIF-2 + homogeneous Co-eIF-2A80 (fraction III) promoted Met-tRNAf.40S complex formation with an AUG codon but not with a physiological mRNA or a polyribonucleotide messenger poly(U,G) whereas eIF-2 + a partially purified Co-eIF-2A80 preparation (fraction II) promoted Met-tRNAf.40S complex formation with an AUG codon as well as with globin mRNA and poly(U,G) messenger. This factor-promoted Met-tRNAf binding to 40S ribosomes depends absolutely on the presence of a polyribonucleotide messenger containing an initiation codon (such as AUG or GUG). Other polyribonucleotide messengers tested, such as poly(U), poly(A) and poly(A,C) were completely ineffective in this binding reaction. This result indicates that the Met-tRNAf.40S.mRNA complex is formed by a direct interaction between Met-tRNAf, 40S ribosomes and the initiation site in mRNA. A mechanism has been proposed for Met-tRNAf.40S.mRNA complex formation in yeast.     

Ribosome-binding sites on chloroplastrbcL andpsbA mRNAs and light-induced initiation of D1 translation

Chloroplast ribosome-binding sites were identified on the plastidrbcL andpsbA mRNAs using toeprint analysis. TherbcL translation initiation domain is highly conserved and contains a prokaryotic Shine-Dalgarno (SD) sequence (GGAGG) located 4 to 12 nucleotides upstream of the initiator AUG. Toeprint analysis ofrbcL mRNA associated with plastid polysomes revealed strong toeprint signals 15 nucleotides downstream from the AUG indicating ribosome binding at the translation initiation site.Escherichia coli 30S ribosomes generated similar toeprint signals when mixed withrbcL mRNA in the presence of initiator tRNA. These results indicate that plastid SD sequences are functional in chloroplast translation initiation. ThepsbA initiator region lacks a SD sequence within 12 nucleotides of the initiator AUG. However, toeprint analysis of soluble and membrane polysome-associatedpsbA mRNA revealed ribosomes bound to the initiator region.E. coli 30S ribosomes did not associate with thepsbA translation initiation region.E. coli and chloroplast ribosomes bind to an upstream region which contains a conserved SD-like sequence. Therefore, translation initiation onpsbA mRNA may involve the transient binding of chloroplast ribosomes to this upstream SD-like sequence followed by scanning to localize the initiator AUG. Illumination 8-day-old dark-grown barley seedlings caused an increase in polysome-associatedpsbA mRNA and the abundance of initiation complexes bound topsbA mRNA. These results demonstrate that light modulates D1 translation initiation in plastids of older dark-grown barley seedlings.     

Molecular Mechanism of Scanning and Start Codon Selection in Eukaryotes

Summary: The correct translation of mRNA depends critically on the ability to initiate at the right AUG codon. For most mRNAs in eukaryotic cells, this is accomplished by the scanning mechanism, wherein the small (40S) ribosomal subunit attaches to the 5′ end of the mRNA and then inspects the leader base by base for an AUG in a suitable context, using complementarity with the anticodon of methionyl initiator tRNA (Met-tRNA_i^Met) as the key means of identifying AUG. Over the past decade, a combination of yeast genetics, biochemical analysis in reconstituted systems, and structural biology has enabled great progress in deciphering the mechanism of ribosomal scanning. A robust molecular model now exists, describing the roles of initiation factors, notably eukaryotic initiation factor 1 (eIF1) and eIF1A, in stabilizing an “open” conformation of the 40S subunit with Met-tRNA_i^Met bound in a low-affinity state conducive to scanning and in triggering rearrangement into a “closed” conformation incompatible with scanning, which features Met-tRNA_i^Met more tightly bound to the “P” site and base paired with AUG. It has also emerged that multiple DEAD-box RNA helicases participate in producing a single-stranded “landing pad” for the 40S subunit and in removing the secondary structure to enable the mRNA to traverse the 40S mRNA-binding channel in the single-stranded form for base-by-base inspection in the P site.     

Unusual ribosome binding properties of mRNA encoding bacteriophage lambda repressor.

The mRNA encoding repressor cI of phage lambda is the only known E. coli message which starts directly with the initiation AUG codon. The ability of in vitro synthesized cI mRNA fragments (150 or 400 nts) to form ternary initiation complexes has been studied using the toeprint method. In the presence of tRNA(Met)f, these fragments are capable of forming the ternary complexes at the 5'-terminal AUG codon not only with 30S subunits but also with undissociated 70S ribosomes (70S tight couples). In the latter case, no binding at other positions of cI mRNA can be detected at all. The starting region of cI mRNA has a single stranded conformation and is highly enriched in A-residues. This feature of cI mRNA RBS is suggested to be the main factor which allows cI mRNA to form the initiation complex with the ribosome. Unlike 30S subunits, the binding to 70S tight couples is not affected by any of the initiation factors, although it is as efficient as that to 30S subunits supplemented with the factors. 30S subunits prefer to associate with the internal RBSs of the preformed mRNA molecules, provided that they are not sequestered by the secondary structure. In contrast, 70S tight couples tend to avoid extra sequences upstream of the codon directed to the P site and occupy a position as close as possible to the 5'-end of the message. This has been found to be the case both for tRNA(Met)f and for elongator tRNA(Glu)2. The structural features of mRNA RBSs which influence their different binding for 30S subunits and 70S ribosomes are discussed.     

Characterization of ribosome binding on Rous sarcoma virus RNA in vitro.

We determined the sites at which ribosomes form initiation complexes on Rous sarcoma virus RNA in order to determine how initiation of Pr76gag synthesis at the fourth AUG codon from the 5' end of Rous sarcoma virus strain SR-A RNA occurs. Ribosomes bind almost exclusively at the 5'-proximal AUG codon when chloride is present as the major anion added to the translational system. However, when chloride is replaced with acetate, ribosomes bind at the two 5'-proximal AUG codons, as well as at the initiation site for Pr76gag. We confirmed that the 5'-proximal AUG codon is part of a functional initiation site by identifying the seven-amino acid peptide encoded there. Our results suggest that (i) translation in vitro of Rous sarcoma virus virion RNA results in the synthesis of at least two polypeptides; (ii) the pattern of ribosome binding observed for Rous sarcoma virus RNA can be accounted for by the modified scanning hypothesis; and (iii) the interaction between 40S ribosomal subunits or 80S ribosomal complexes is stronger at the 5'-proximal AUG codon than at sites farther downstream, including the initiation site for the major viral proteins.     

Context effects and inefficient initiation at non-AUG codons in eucaryotic cell-free translation systems.

The context requirements for recognition of an initiator codon were evaluated in vitro by monitoring the relative use of two AUG codons that were strategically positioned to produce long (pre-chloramphenicol acetyl transferase [CAT]) and short versions of CAT protein. The yield of pre-CAT initiated from the 5'-proximal AUG codon increased, and synthesis of CAT from the second AUG codon decreased, as sequences flanking the first AUG codon increasingly resembled the eucaryotic consensus sequence. Thus, under prescribed conditions, the fidelity of initiation in extracts from animal as well as plant cells closely mimics what has been observed in vivo. Unexpectedly, recognition of an AUG codon in a suboptimal context was higher when the adjacent downstream sequence was capable of assuming a hairpin structure than when the downstream region was unstructured. This finding adds a new, positive dimension to regulation by mRNA secondary structure, which has been recognized previously as a negative regulator of initiation. Translation of pre-CAT from an AUG codon in a weak context was not preferentially inhibited under conditions of mRNA competition. That result is consistent with the scanning model, which predicts that recognition of the AUG codon is a late event that occurs after the competition-sensitive binding of a 40S ribosome-factor complex to the 5' end of mRNA. Initiation at non-AUG codons was evaluated in vitro and in vivo by introducing appropriate mutations in the CAT and preproinsulin genes. GUG was the most efficient of the six alternative initiator codons tested, but GUG in the optimal context for initiation functioned only 3 to 5% as efficiently as AUG. Initiation at non-AUG codons was artifactually enhanced in vitro at supraoptimal concentrations of magnesium.     

The sequence context of the initiation codon in the encephalomyocarditis virus leader modulates efficiency of internal translation initiation.

Translation initiation on poliovirus and encephalomyocarditis virus (EMCV) mRNAs occurs by a cap-independent mechanism utilizing an internal ribosomal entry site (IRES). However, no unifying mechanism for AUG initiation site selection has been proposed. Analysis of initiation of mRNAs translated in vitro has suggested that initiation of poliovirus mRNA translation likely involves both internal binding of ribosomes and scanning to the first AUG which is in a favorable context for initiation. In contrast, internal initiation on EMCV mRNA may not utilize scanning, since ribosomes bind directly or very close to the initiation codon AUG-11. We have studied in vivo the sequence requirements for internal initiation around the EMCV initiation codon, both in monocistronic and in dicistronic mRNAs. Our studies show that the upstream AUG-10 is normally not used and that there is no specific sequence requirement for nucleotides between AUG-10 and AUG-11. However, the sequence context of AUG-11 does influence the efficiency of initiation at AUG-11. Efficient IRES-mediated internal initiation at AUG-11 exhibits a requirement for an adenine in the -3 position, similar to cap-dependent initiation. These results support a model for internal initiation on EMCV mRNA in which scanning starts at or near AUG-11. Although initiation primarily occurs at AUG-11, initiation at multiple downstream AUG codons can be detected. In addition, a poor sequence context around AUG-11 results in increased initiation at one or more downstream AUG codons, indicative of leaky scanning or jumping by the ribosome from AUG-11 mediated by the EMCV IRES.     

Stage-specific initiation factors for protein synthesis during insect development

In insects, as in bacteria, the smaller (40 S) ribosomal subunit binds messenger RNA during initiation of protein synthesis. An 80 S ribosomal unit is formed by association of free 40 S and 60 S subunits. Formation of the complete initiation complex requires GTP, aminoacyl-tRNA, protein initiation factors and messenger RNA. The complex sediments as an 80 S band on sucrose gradient. Protein initiation factors are extracted from unwashed ribosomes and appear to be able to discriminate between messenger RNAs obtained from different stages of development. They promote formation of the 80 S complex only when messenger RNA is extracted from the same stage of development, providing a mechanism for control of protein synthesis by which ribosomes can select the messenger RNA to be translated. Two possibilities have been proposed to explain this phenomenon: (1) that a group of messenger RNAs from a given stage of development may have a specific sequence of nucleotides preceding the AUG codon. This sequence is recognized by a stage-specific element of the initiation machinery; (2) and or, the secondary structure of messenger RNA from a given stage of development may be specific and therefore recognized by a unique initiation factor.     

Ribosome-initiator tRNA complex as an intermediate in translation initiation in Escherichia coli revealed by use of mutant initiator tRNAs and specialized ribosomes.

For functional studies of mutant Escherichia coli initiator tRNAs in vivo, we previously described a strategy based on the use of tRNA genes carrying an anticodon sequence change from CAU to CUA along with a mutant chloramphenicol acetyltransferase (CAT) gene carrying an initiation codon change from AUG to UAG. Surprisingly, under conditions where the mutant initiator tRNA is optimally active, the CAT gene with the UAG initiation codon produced more CAT protein (3- to 9-fold more depending on the conditions) than the wild-type CAT gene. Here we show that two new mutant CAT genes having GUC and AUC initiation codons also produce more of the CAT protein in the presence of the corresponding mutant initiator tRNAs. These results are most easily understood if assembly of the 30S ribosome-initiator tRNA-mRNA initiation complex in vivo proceeds with the 30S ribosome binding first to the initiator tRNA and then to the mRNA. In cells overproducing the mutant initiator tRNAs, most ribosomes would carry the mutant initiator tRNA and these ribosomes would select the mutant CAT mRNA over the other mRNAs.