How do eukaryotic ribosomes recognize the unique AUG initiator codon in messenger RNA?
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Base Sequence↗
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Identification of the ATG initiation codon in human type I desmocollin.
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Base Sequence↗
Mutation of a termination codon affects src initiation.
The four Rous sarcoma virus messages gag, gag-pol, env, and src all derive from a full-length RNA precursor. All four messages contain the same 5' leader segment. Three of the messages, gag, gag-pol, and env, use an AUG present in this leader to initiate translation. The src AUG initiation codon lies 3' of the leader segment, 90 bases downstream of the gag initiation codon in the spliced src message. However, in the spliced src message a UGA termination codon lies between the gag AUG and the src AUG. All three codons are in the same reading frame. By using oligonucleotide-directed mutagenesis, the UGA termination codon has been converted to CGA. Cells infected with the mutant (called 1057 CGA) were spindle shaped, distinct from the rounded shape of cells infected with the parental Rous sarcoma virus. The mutant virus initiates src translation at the gag AUG, producing a 63,000-dalton src protein. We suggest that the wild-type src message produces two polypeptides, a very small (nine-amino acid) peptide that is initiated at the gag AUG and the 60,000-dalton src protein that is initiated at the src AUG.
Amino Acid Sequence↗
Regulation of mdm2 expression by p53: alternative promoters produce transcripts with nonidentical translation potential.
The mdm2 proto-oncogene product binds to the p53 tumor suppressor protein and inhibits its ability to trans-activate target genes. One such target gene is mdm2 itself, which is therefore considered a component of a p53 negative feedback loop. Two tandem p53-binding motifs residing within the first intron of the murine mdm2 gene confer upon it p53-mediated activation. We now report that in murine cells p53 activates an internal mdm2 promoter (P2) located near the 3' end of intron 1, resulting in mRNA whose transcription starts within exon 2. P2 is activated by p53 within artificial constructs, as well as within the context of the chromosomal mdm2 gene. Activation follows either the introduction of overexpressed wild-type p53 into cells or the induction of endogenous wild-type p53 by ionizing radiation. The upstream, constitutive (P1) mdm2 promoter is only mildly affected by p53, if at all. The p53-derived mdm2 transcripts lack exon 1 and a few nucleotides from exon 2. As the first in-frame AUG of mdm2 is located within exon 3, the two types of mdm2 transcripts should possess similar coding potentials. Nevertheless, in vitro conditions, where each of these transcripts yields a distinct translation profile, reflect the differential usage of translation initiation codons. Initiation of translation at internal AUG codons, which occurs also in vivo, gives rise to MDM2 polypeptides incapable of binding to p53. In vitro translation profiles of the various mdm2 transcripts could be manipulated by changing the amounts of input RNA. Thus, p53 can modulate both the amount and the nature of MDM2 polypeptides through activation of the internal P2 promoter.
3T3 Cells↗
The suil suppressor locus in Saccharomyces cerevisiae encodes a translation factor that functions during tRNA(iMet) recognition of the start codon.
We initiated a genetic reversion analysis at the HIS4 locus to identify components of the translation initiation complex that are important for ribosomal recognition of an initiator codon. Three unlinked suppressor loci, suil, sui2, and SUI3, that restore expression of both HIS4 and HIS4-lacZ in the absence of an AUG initiator codon were identified. In previous studies, it was demonstrated that the sui2 and SUI3 genes encode mutated forms of the alpha and beta subunits, respectively, of eukaryotic translation initiation factor 2 (eIF-2). In this report, we describe the molecular and biochemical characterizations of the sui1 suppressor locus. The DNA sequence of the SUI1+ gene shows that it encodes a protein of 108 amino acids with a calculated Mr of 12,300. The sui1 suppressor genes all contain single base pair changes that alter a single amino acid within this 108-amino-acid sequence. sui1 suppressor strains that are temperature sensitive for growth on enriched medium have altered polysome profiles at the restrictive temperature typical of those caused by alteration of a protein that functions during the translation initiation process. Gene disruption experiments showed that the SUI1+ gene encodes an essential protein, and antibodies directed against the SUI1+ coding region identified a protein with the predicted Mr in a ribosomal salt wash fraction. As observed for sui2 and SUI3 suppression events, protein sequence analysis of His4-beta-galactosidase fusion proteins produced by sui1 suppression events indicated that a UUG codon is used as the site of translation initiation in the absence of an AUG start codon in HIS4. Changing the penultimate proline codon 3' to UUG at his4 to a Phe codon (UUC) blocks aminopeptidase cleavage of the amino-terminal amino acid of the His4-beta-galactosidase protein, as noted by the appearance of Met in the first cycle of the Edman degradation reaction. The appearance of Met in the first cycle, as noted, in either a sui1 or a SUI3 suppressor strain showed that the mechanism of suppression is the same for both suppressor genes and allows the initiator tRNA to mismatch base pair with the UUG codon. This suggests that the Sui1 gene product performs a function similar to that of the beta subunit of eIF-2 as encoded by the SUI3 gene. However, the Sui1 gene product does not appear to be a required subunit of eIF-2 on the basis of purification schemes designed to identify the GTP-dependent binding activity of eIF-2 for the initiator tRNA. In addition, suppressor mutations in the sui1 gene, in contrast to suppressor mutations in the sui2 or SUI3 gene, do not alter the GTP-dependent binding activity of the eIF-2. The simplest interpretation of these studies is that the sui1 suppressor gene defines an additional factor that functions in concert with eIF-2 to enable tRNAiMet to establish ribosomal recognition of an AUG initiator codon.
Amino Acid Sequence↗
In vivo evidence for the prokaryotic model of extended codon-anticodon interaction in translation initiation.
Initiation codon context is an important determinant of translation initiation rates in both prokaryotes and eukaryotes. Such sequences include the Shine- Dalgarno ribosome-binding site, as well as other motifs surrounding the initiation codon. One proposed interaction is between the base immediately preceding the initiation codon (-1 position) and the nucleotide 3' to the tRNAf(Met) anticodon, at position 37. Adenine is conserved at position 37, and a uridine at -1 has been shown in vitro to favor initiation. We have tested this model in vivo, by manipulating the chloroplast of the green alga Chlamydomonas reinhardtii, where the translational machinery is prokaryotic in nature. We show that translational defects imparted by mutations at the petA -1 position can be suppressed by compensatory mutations at position 37 of an ectopically expressed tRNA(fMet). The mutant tRNAs are fully aminoacylated and do not interfere with the translation of other proteins. Although this extended base pairing is not an absolute requirement for initiation, it may convey added specificity to transcripts carrying non-standard initiation codons, and/or preserve translational fidelity under certain stress conditions.
Algal Proteins↗
Ribosomal proteins cross-linked to the initiator AUG codon of a mRNA in the translation initiation complex by UV-irradiation.
Eukaryotic ribosomal proteins constituting the binding site for the initiator codon AUG on the ribosome at the translation initiation step were investigated by UV-induced cross-linking between protein and mRNA. The 80S-initiation complex was formed in a rabbit reticulocyte cell-free system in the presence of sparsomycin with radiolabeled Omega-fragment as a template, which was a 73-base 5'-leader sequence of tobacco mosaic virus RNA having AUG at the extreme 3'-terminal end and extended with 32pCp. Two radioactive peaks were sedimented by sucrose gradient centrifugation, one being the 80S initiation complex formed at the 3'-terminal AUG codon, and the other presumably a "disome" with an additional 80S ribosome bound at an upstream AUU codon, formed when Omega-fragment was incubated with sparsomycin [Filipowicz and Henni (1979) Proc. Natl. Acad. Sci. USA 76, 3111-3115]. Cross-links between ribosomal proteins and the radiolabeled Omega-fragment were induced in situ by UV-irradiation at 254 nm. After extensive nuclease digestion of the complexes, ribosomal proteins were separated by two-dimensional gel electrophoresis. Autoradiography identified the proteins S7, S10, S25, S29, and L5 of the 80S initiation complex and S7, S25, S29 and L5 of that in the disome as 32P-labeled proteins. Together with the results of cross-linking experiments of other investigators and recently solved crystal structures of prokaryotic ribosomes, the spatial arrangement of eukaryotic ribosomal proteins at the AUG-binding domain is discussed.
Animals↗
Translation of chloroplast psbD mRNA in Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start codon.
Translation initiation represents a key step during regulation of gene expression in chloroplasts. Here, we report on the identification and characterization of three suppressor point mutations which overcome a translational defect caused by the deletion of a U-rich element in the 5'-untranslated region (5'-UTR) of the psbD mRNA in the green alga Chlamydomonas reinhardtii. All three suppressors affect a secondary RNA structure encompassing the psbD AUG initiation codon within a double-stranded region as judged by the analysis of site-directed chloroplast mutants as well as in vitro RNA mapping experiments using RNase H. In conclusion, the data suggest that these new element serves as a negative regulator which mediates a rapid shut-down of D2 synthesis.
5' Untranslated Regions↗
Translation of Sindbis virus mRNA: analysis of sequences downstream of the initiating AUG codon that enhance translation.
Alphaviruses, particularly Sinbis virus and Semliki Forest virus, are proving to be useful vectors for the expression of heterologous genes. In infected cells, these self-replicating vectors (replicons) transcribe a subgenomic mRNA that codes for a heterologous protein instead of the structural proteins. We reported recently that translation of the reporter gene lacZ is enhanced 10-fold when the coding sequences of this gene are fused downstream of and in frame with the 5' half of the capsid gene (I. Frolov and S. Schlesinger, J. Virol. 68:8111-8117, 1994). The enhancing sequences, located downstream of the AUG codon that initiates translation of the capsid protein, have a predicted hairpin structure. We have mutated this region by making changes in the codons which do not affect the protein sequence but should destabilize the putative hairpin structure. These changes caused a decrease in the accumulation of the capsid-beta-galactosidase fusion protein. When these alterations were inserted into the capsid gene in the context of the intact Sindbis virus genome, they led to a decrease in the rate of virus formation but did not affect the final yield. We also altered the original sequence to one that has 12 contiguous G.C base pairs and should form a stable hairpin. The new sequence was essentially as effective as the original had been in enhancement of translation and in the rate of virus formation. The position of the predicted hairpin structure is important for its function; an insertion of 9 nucleotides or a deletion of 9 nucleotides decreased the level of translation. The insertion of a hairpin structure at a particular location downstream of the initiating AUG appears to be a way that alphaviruses have evolved to enhance translation of their mRNA, and, as a consequence, they produce high levels of the structural proteins which are needed for virus assembly. This high level of translation requires an intracellular environment in which host cell protein synthesis is inhibited.
Amino Acid Sequence↗
Expression of the Escherichia coli pcnB gene is translationally limited using an inefficient start codon: a second chromosomal example of translation initiated at AUU.
Expression of the gene pcnB, encoding the dispensable Escherichia coli poly(A) polymerase (PAPI), which is toxic when overproduced, was investigated. Its promoter was identified and found to be moderately strong when used to express a beta-galactosidase reporter. Expression levels were not affected by increasing or decreasing PcnB concentration. Translation of pcnB was found to initiate from the non-canonical initiation codon AUU. The only other coli gene reported to use AUU as initiation codon is infC, which encodes the initiation factor IF-3. AUU, in common with other rarely used initiation codons, is discriminated against by IF-3, resulting in the aborting of most AUU-promoted initiation events. This enables AUU to form part of an autoregulatory circuit controlling IF-3 production. We show that InfC discrimination reduces PcnB production fivefold. This is the first instance of this mechanism being used to limit severely the production of a potentially toxic product.
Base Sequence↗
Role of the AGA/AGG codons, the rarest codons in global gene expression in Escherichia coli.
AGA and AGG codons for arginine are the least used codons in Escherichia coli. Previous findings have shown that these codons are used preferentially within the first 25 codons in E. coli genes. More than 100 genes having a single AGA/AGG codon within the first 25 codons were identified to be associated with various essential cellular functions. The lacZ gene, containing 5 AGG codons after the tenth codon from the initiation codon, was constructed as a model system. The production of beta-galactosidase was inhibited almost completely during the stationary phase, whereas the production of the control beta-galactosidase without AGG codons was not. The inhibitory effect by the 5 AGG codons was substantially suppressed either by coexpressing the argU gene for tRNA(ArgUCU/CCU) or by moving the 5 AGG codons by > 50 codons away from the initiation codon. In addition, the production of a number of proteins resolved by two-dimensional gel electrophoresis was enhanced significantly during the stationary phase in the cells harboring a plasmid containing argU. At least one of them was identified as the hns product encoded by an ORF having an AGA codon at the nineteenth position. On the basis of these results, it is proposed that the expression of a group of essential genes for various cellular functions that have a single AGA/AGG codon very close to the initiation codon are globally regulated by the availability of the least abundant tRNA(ArgUCU/CCU). A model for this regulation is proposed.
Arginine↗
Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus beta-lactamase gene.
The base sequence of the ribosome binding site region of the Gram-positive Staphylococcus aureus beta-lactamase gene has been determined. The leader peptide sequence of 24 amino acids which precedes the NH2 terminus of extracellular S. aureus beta-lactamase has also been established. This initiation site possesses two unique features not observed for most initiation sites recognized by Escherichia coli ribosomes. A novel initiation codon, UUG, initiates protein synthesis with methionine; and a very strong Shine-Dalgarno complementarity containing five G-C base pairs precedes the UUG initiation codon. The strong Shine-Dalgarno complementarity may explain the reduced translational dependence on initiation factor IF-3 function that has been observed for the beta-lactamase mRNA and other mRNAs from Gram-positive bacteria. We suggest that this extent of complementarity between the mRNA and this extent of complementarity between the mRNA and the ribosome may be a requirement for efficient initiation by Bacillus subtilis and other Gram-positive ribosomes, and may provide the basis for the observed inability of the Gram-positive systems to translate most of the mRNAs from Gram-negative bacteria.
Amino Acid Sequence↗
Homozygous initiation codon-altering complex variant causes rapid-onset chorioretinopathy phenotype in ABCA4 disease.
PURPOSE: To characterize the clinical phenotype associated with a homozygous start codon-altering complex variant in the ABCA4 gene and evaluate its severity and prognosis in the context of Stargardt disease. METHODS: Patient records were retrospectively reviewed for homozygous ABCA4 start codon variants. Patients underwent ophthalmic exam, multimodal imaging, full-field electroretinography (ffERG), and inherited retinal disease panel testing. Structural and functional retinal assessments were reviewed to determine phenotype severity. RESULTS: Three brothers of Ashkenazi Jewish descent presented with profound early-onset vision loss beginning at age 7, with best-corrected visual acuity reduced to counting fingers or hand motion by early adulthood. Imaging revealed widespread macular atrophy, extensive intraretinal pigment migration, and near-complete foveal outer nuclear layer loss. ffERG demonstrated extinguished scotopic and photopic responses. The patients were found to be homozygous for a complex ABCA4 allele containing the start codon variant c.[1A > G;6089G > A]. These features were consistent with the rapid-onset chorioretinopathy phenotype, previously associated with null ABCA4 alleles. CONCLUSIONS: This report characterizes the clinical findings in patients homozygous for the c.[1A > G;6089G > A] variant in ABCA4, confirming its association with a severe, rapid-onset chorioretinopathy phenotype. The data support the pathogenic nature of this complex allele and expands the genotype-phenotype correlational spectrum of ABCA4-related disease, with implications for prognosis and genetic counseling.
Humans↗
The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo.
A method is described to determine simultaneously the effect of any changes in the ribosome-binding site (RBS) of mRNA on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. The approach was used to analyse systematically the influence of spacing between the Shine-Dalgarno sequence and the initiation codon, the three different initiation codons, and RBS secondary structure on translational yields in the two organisms. Both B. subtilis and E. coli exhibited similar spacing optima of 7-9 nucleotides. However, B. subtilis translated messages with spacings shorter than optimal much less efficiently than E. coli. In both organisms, AUG was the preferred initiation codon by two- to threefold. In E. coli GUG was slightly better than UUG while in B. subtilis UUG was better than GUG. The degree of emphasis placed on initiation codon type, as measured by translational yield, was dependent on the strength of the Shine-Dalgarno interaction in both organisms. B. subtilis was also much less able to tolerate secondary structure in the RBS than E. coli. While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity. The approach described provides a rapid and systematic means of elucidating such additional determinants.
Bacillus subtilis↗
Suppression of the negative effect of minor arginine codons on gene expression; preferential usage of minor codons within the first 25 codons of the Escherichia coli genes.
AGA and AGG codons for arginine are the least used codons in Escherichia coli, which are encoded by a rare tRNA, the product of the dnaY gene. We examined the positions of arginine residues encoded by AGA/AGG codons in 678 E. coli proteins. It was found that AGA/AGG codons appear much more frequently within the first 25 codons. This tendency becomes more significant in those proteins containing only one AGA or AGG codon. Other minor codons such as CUA, UCA, AGU, ACA, GGA, CCC and AUA are also found to be preferentially used within the first 25 codons. The effects of the AGG codon on gene expression were examined by inserting one to five AGG codons after the 10th codon from the initiation codon of the lacZ gene. The production of beta-galactosidase decreased as more AGG codons were inserted. With five AGG codons, the production of beta-galactosidase (Gal-AGG5) completely ceased after a mid-log phase of cell growth. After 22 hr induction of the lacZ gene, the overall production of Gal-AGG5 was 11% of the control production (no insertion of arginine codons). When five CGU codons, the major arginine codon were inserted instead of AGG, the production of beta-galactosidase (Gal-CGU5) continued even after stationary phase and the overall production was 66% of the control. The negative effect of the AGG codons on the Gal-AGG5 production was found to be dependent upon the distance between the site of the AGG codons and the initiation codon. As the distance was increased by inserting extra sequences between the two codons, the production of Gal-AGG5 increased almost linearly up to 8 fold. From these results, we propose that the position of the minor codons in an mRNA plays an important role in the regulation of gene expression possibly by modulating the stability of the initiation complex for protein synthesis.
Amino Acid Sequence↗
Structural and functional relationships between fumarase and aspartase. Nucleotide sequences of the fumarase (fumC) and aspartase (aspA) genes of Escherichia coli K12.
The nucleotide sequences of two segments of DNA (2250 and 2921 base-pairs) containing the functionally related fumarase (fumC) and aspartase (aspA) genes of Escherichia coli K12 were determined. The fumC structural gene comprises 1398 base-pairs (466 codons, excluding the initiation codon), and it encodes a polypeptide of Mr 50353 that resembles the fumarases of Bacillus subtilis 168 (citG-gene product), rat liver and pig heart. The fumC gene starts 140 base-pairs downstream of the structurally-unrelated fumA gene, but there is no evidence that both genes form part of the same operon. The aspA structural gene comprises 1431 base-pairs (477 codons excluding the initiation codon), and it encodes a polypeptide of Mr 52190, similar to that predicted from maxicell studies and for the enzyme from E. coli W. Remarkable homologies were found between the primary structures of the fumarase (fumC and citG) and aspartase (aspA) genes and their products, suggesting close structural and evolutionary relationships.
Amino Acid Sequence↗
Low conservation of functional domains of HIV type 1 vif and vpr genes in infected mothers correlates with lack of vertical transmission.
Human immunodeficiency virus type 1 (HIV-1) vif and vpr sequences were analyzed from four nontransmitting mothers (infected mothers who failed to transmit HIV-1 to their infants mainly in the absence of anti-retroviral therapy), including a mother with multiple deliveries, and compared with the vif and vpr sequences of five and six previously analyzed transmitting mothers, respectively. In contrast to a high functional conservation of vif and vpr genes in transmitting mother isolates, we found that there was a low degree of conservation of functional domains of these genes in nontransmitting (NT) mother isolates. For vif sequences, NT-2 contained stop codons and no initiation codons, whereas NT-1 sequences carried a substitution of a highly conserved tyrosine to histidine at position 30. In addition, NT-3 and NT-4 sequences contained additional substitutions, including asparagine at position 22, lysine at position 77 and histidine at position 110, that were absent in transmitting mother and consensus subtype B sequences. Similarly, the vpr sequences of NT-2 contained stop codons and no initiation codons, NT-4 contained a substitution of serine in place of alanine at position 30, some NT-1 sequences substituted arginine in place of glycine at position 75, and NT-3 sequences presented a deletion in the C terminus that was absent in transmitting mother and consensus subtype B sequences and is essential for Vpr function. Furthermore, vif and vpr sequences of nontransmitting mothers were less heterogeneous compared with transmitting mother sequences. In conclusion, a low degree of conservation of functional domains and heterogeneity of HIV-1 vif and vpr genes in these infected mothers correlates with lack of vertical transmission.
Adult↗
Characteristic distribution of bases and codons around the initiation and termination codons in whole reading frames in bacteria and yeast genomes.
Recently the complete nucleotide sequence of the entire genome was determined for yeast and a few kinds of bacterium. To see characteristic features of base sequence in the cistron (actually the open reading frame, ORF) and in the regions around a cistron (ORF), the biases of appearance frequency of bases from the base ratio were studied statistically. In the regions before the base biases were observed. The characteristic base distribution patterns were similar to all the cases of bacteria, but different from yeast. The base biases are reflected on the appearance frequency of amino acids near the N-termini and C-termini of proteins. The characteristic biases found in the amino acid sequence of the N-terminal part of bacteria proteins are different from that in yeast proteins.
Bacillus subtilis↗