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PubMed2006

Where to begin? The mechanism of translation initiation codon selection in eukaryotes.

Selecting the codon at which to begin translation is a complicated event in an already complicated process. Many protein initiation factors (eIFs) have been implicated in start site selection, but the mechanistic details of their activities have remained obscure until recently. Biochemical and genetic studies of eIFs 1, 1A, 2 and 5 have suggested that the 43S pre-initiation complex exists in two conformations and that the changing interactions of the factors within the 43S pre-initiation complex in response to encountering an AUG codon regulates these conformations and, ultimately, the selection of the start codon.

Animals↗
PubMed1989

An initiation codon mutation in the apoC-II gene (apoC-II Paris) of a patient with a deficiency of apolipoprotein C-II.

We have identified the genetic defect that leads to a deficiency of apoC-II in the proband from the Paris kindred. Analysis of the apoC-IIParis DNA by Southern blot hybridization revealed no major gene rearrangements, but sequencing of polymerase chain reaction-amplified apoC-IIParis DNA revealed an A to G transition that changed the initiation AUG (methionine) codon to GUG (valine). Potential initiation of translation at the closest inframe methionine codon eliminates the entire signal peptide and the first 8 amino-terminal residues of apoC-II which would prevent apoC-II secretion into plasma. In agreement with this, no apoC-II was detected in the patient's plasma by radioimmunoassay or by two-dimensional gel electrophoresis and immunoblotting. Direct sequencing of amplified patient DNA from 12 different polymerase chain reaction samples demonstrated the presence of the A to G substitution in all, indicating that the proband is a homozygote for the defect. We propose that in the apoC-IIParis gene, a mutation in the initiation methionine codon prevents the normal initiation of apolipoprotein synthesis and leads to a deficiency of apoC-II. This initiation methionine mutation represents a new type of molecular defect that can result in Type I hyperlipoproteinemia.

Adult↗
PubMed2004

Nonsense mutations in close proximity to the initiation codon fail to trigger full nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs containing premature translation termination codons. In mammalian cells, a termination codon is ordinarily recognized as "premature" if it is located greater than 50-54 nucleotides 5' to the final exon-exon junction. We have described a set of naturally occurring human beta-globin gene mutations that apparently contradict this rule. The corresponding beta-thalassemia genes contain nonsense mutations within exon 1, and yet their encoded mRNAs accumulate to levels approaching wild-type beta-globin (beta(WT)) mRNA. In the present report we demonstrate that the stabilities of these mRNAs with nonsense mutations in exon 1 are intermediate between beta(WT) mRNA and beta-globin mRNA carrying a prototype NMD-sensitive mutation in exon 2 (codon 39 nonsense; beta 39). Functional analyses of these mRNAs with 5'-proximal nonsense mutations demonstrate that their relative resistance to NMD does not reflect abnormal RNA splicing or translation re-initiation and is independent of promoter identity and erythroid specificity. Instead, the proximity of the nonsense codon to the translation initiation AUG constitutes a major determinant of NMD. Positioning a termination mutation at the 5' terminus of the coding region blunts mRNA destabilization, and this effect is dominant to the "50-54 nt boundary rule." These observations impact on current models of NMD.

Animals↗
PubMed2000

The pathogenic role of point mutations affecting the translational initiation codon of mitochondrial genes.

The mutation T3308C results in a Met --> Thr change at the highly conserved amino acid position 1 of the mtDNA ND1 gene (M1T). To study its potential pathogenic effect we have carried out a combination of mitochondrial protein synthesis and Northern and Western analyses. Our data demonstrate that M1T mutation does not affect the efficiency of the synthesis of the ND1 polypeptide and suggest that any codon specifying methionine located close to the 5' end of mitochondrial mRNAs may be used as translational initiator.

Blotting, Northern↗
PubMed2003

Identification of a novel internal ribosome entry site in giardiavirus that extends to both sides of the initiation codon.

In Giardia lamblia, enhanced translation of luciferase mRNA, flanked between the 5'-untranslated region (UTR) and 3 '-end of giardiavirus transcript, requires the presence of the initial 264-nucleotide (nt) viral capsid-coding region. By introducing the transcripts of dicistronic viral constructs into Giardia, we demonstrated that the 264-nt downstream region alone is insufficient to function as an internal ribosome entry site (IRES) without including a portion of the 5 '-UTR as well. Deletion analysis showed that efficient internal initiation requires the last 253 nts (nts 114-367) of the 5 '-UTR in combination with the downstream 264 nts. Specific mutations that disrupted the predicted secondary structural elements in either the 5 '-UTR or the 264-nt capsid-coding region completely abolished the IRES-mediated translation of downstream cistron, suggesting that the IRES activity requires the presence of these structures in both regions. Mutations that abolished translation of the first cistron did not, however, affect the IRES-mediated translation of the second cistron, indicating that this IRES-mediated translation is independent of the translation of the upstream cistron. This is, to our knowledge, the first reported identification of a viral IRES with an estimated size of 517 nts that extends to both sides of the initiation site.

5' Untranslated Regions↗
PubMed1997

Internal entry of ribosomes is directed by the 5' noncoding region of classical swine fever virus and is dependent on the presence of an RNA pseudoknot upstream of the initiation codon.

Bicistronic RNAs containing the 373-nucleotide-long 5' nontranslated region (NTR) of the classical swine fever virus (CSFV) genome as intercistronic spacer were used to show the presence of an internal ribosome entry site (IRES) in the 5' end of the CSFV genome. By coexpression of the poliovirus 2A protease it was demonstrated that the CSFV 5' NTR-driven translation is independent of the presence of functional eukaryotic initiation factor eIF-4F. Deletion analysis indicated that the 5' border of the IRES is located between nucleotides 28 and 66. The role of a proposed pseudoknot structure at the 3' end of the CSFV 5' NTR in IRES-mediated translation was investigated by site-directed mutagenesis. Mutant RNAs that had lost the ability to base pair in stem II of the pseudoknot were translationally inactive. Translation to wild-type levels could be restored through the introduction of compensatory complementary base changes that repaired base pairing in stem II. In addition, we showed that the AUG codon, which is located 7 nucleotides upstream of the polyprotein initiation site and is conserved in pestiviruses, could not be used to initiate translation. Also, an AUG codon introduced downstream of the polyprotein initiation site was not recognized as an initiation site by ribosomes. These data suggest that after internal entry on the CSFV 5' NTR, ribosomal scanning for the initiation codon is limited to a small region.

Animals↗
PubMed1994

Positions +5 and +6 can be major determinants of the efficiency of non-AUG initiation codons for protein synthesis.

Only rarely do GUG (or CUG or ACG) codons which precede the 5'-proximal AUG function as initiators of protein synthesis, even when they are within a context that contains a purine at position -3 and a G at +4. For example, the upstream GUG of the human parainfluenza virus type 1 (hPIV1) P gene is initiated by ribosomes at high frequency, whereas a seemingly similar GUG codon in the hPIV3 P gene is not used at all. We have examined the reasons for this by expressing chimeric hPIV3/hPIV1 mRNAs, both in vivo and in vitro. A major determinant for efficient GUG utilization was located downstream of the GUG, but this did not appear to be involved in the formation of secondary structure. Rather, the sequence immediately downstream was found to be critical; this determinant was mapped to positions +5 and +6. GUG could be used efficiently for ribosomal initiation when the second codon was GAU but not when it was GUA. Similar results were found when other non-AUG start sites, the Sendai virus P gene ACG and the c-myc-1 CUG, were examined. These results suggest that positions +5 and +6 are important determinants for initiation at non-AUG start sites, and that they are recognized independently of the overall secondary structure of the mRNA.

Base Sequence↗
PubMed1992

Both forms of translational initiation factor IF2 (alpha and beta) are required for maximal growth of Escherichia coli. Evidence for two translational initiation codons for IF2 beta.

The gene infB codes for two forms of translational initiation factor IF2; IF2 alpha (97,300 Da) and IF2 beta (79,700 Da). IF2 beta arises from an independent translational event on a GUG codon located 471 bases downstream from IF2 alpha start codon. By site-directed mutagenesis we constructed six different mutations of this GUG codon. In all cases, IF2 beta synthesis was variably affected by the mutations but not abolished. We show that the residual expression of IF2 beta results from translational initiation on an AUG codon located 21 bases downstream from the mutated GUG. Furthermore, two forms of IF2 beta have been separated by fast protein liquid chromatography and the determination of their N-terminal sequences indicated that they resulted from two internal initiation events, one occurring on the previously identified GUG start codon, the other on the AUG codon immediately downstream. We conclude that two forms of IF2 beta exist in the cell, which differ by seven aminoacid residues at their N terminus. Only by mutating both IF2 beta start codons could we construct plasmids that express only IF2 alpha. A plasmid expressing only IF2 beta was obtained by deletion of the proximal region of the infB gene. Using a strain that carries a null mutation in the chromosomal copy of infB and a functional copy of the same gene on a thermosensitive lysogenic lambda phage, we could cure the lambda phage when the plasmids expressing only one form of IF2 were supplied in trans. We found that each one of the two forms of IF2, at near physiological levels, can support growth of Escherichia coli, but that growth is retarded at 37 degrees C. This result shows that both forms of IF2 are required for maximal growth of the cell and suggests that they have acquired some specialized but not essential function.

Amino Acid Sequence↗
PubMed1997

A dominant mutation in the Chlamydomonas reinhardtii nuclear gene SIM30 suppresses translational defects caused by initiation codon mutations in chloroplast genes.

A suppressor of a translation initiation defect caused by an AUG to AUU mutation in the Chlamydomonas reinhardtii chloroplast petD gene was isolated, defining a nuclear locus that we have named SIM30. A dominant mutant allele at this locus, sim30-1d, was found to increase the translation initiation rate of the mutant petD mRNA. sim30-1d was also able to suppress the translational defect caused by an AUG to AUC mutation in the petD gene, and an AUG to AUU mutation in the chloroplast petA gene. We therefore suggest that the SIM30 gene may encode a general chloroplast translation factor. The ability of sim30-1d to suppress the petD AUG to AUU mutation is diminished in the presence of one or more antibiotic resistance markers located within the 16S and 23S rRNAs, suggesting that the activity of the sim30-1d gene product in translation initiation may involve interaction with ribosomal subunits.

Animals↗
PubMed1983

The beta2-microglobulin mRNA in human Daudi cells has a mutated initiation codon but is still inducible by interferon.

The human Burkitt lymphoma cell line Daudi does not synthesize beta2-microglobulin (beta2m) and lacks the cell surface histocompatibility antigens. The cells, however, contain RNA hybridizing to a cloned human beta2m cDNA probe. cDNA from this Daudi beta2m RNA, was cloned and sequenced. By comparison with cDNA prepared from Ramos cells, which synthesized microglobulin, we determined the sequence of the 20 amino acid long leader peptide of pre-beta2m and show that in Daudi cells the initiator ATG has been mutated to ATC. Although Daudi beta2m RNA cannot be translated, interferon induces the beta2m RNA in Daudi cells as well as in normal human cells.

Amino Acid Sequence↗
PubMed1971

The leader sequence from the 5'-terminus to the A-protein initiation codon in MS2-virus RNA.

RNA fragments of different chain length, each containing the 5'-terminal guanosine tetraphosphate (pppGp) of bacteriophage-MS2 RNA, have been isolated from partial ribonuclease digests of the viral RNA. The longest fragment overlaps with the ribosomalbinding site of the A-protein cistron. The base sequence has been established for the major part. The results directly confirm that the A-protein cistron is closest to the 5'-terminus. Its initiating (AUG) codon starts at position 130, being preceded by an untranslated sequence of 129 nucleotides.

Autoradiography↗
PubMed1982

Eukaryotic ribosomes can recognize preproinsulin initiation codons irrespective of their position relative to the 5' end of mRNA.

The functional assay of a eukaryotic mRNA, into which additional AUG codons have been introduced by in vitro mutagenesis, shows that a translational initiation site need not necessarily be the nearest AUG codon to the 5' end of a mRNA. Hence, the sequence surrounding the AUG, and not simply its position relative to the 5' end of mRNA, appears to be important in determining initiation efficiency.

Animals↗
PubMed1988

An initiator codon mutation in ornithine-delta-aminotransferase causing gyrate atrophy of the choroid and retina.

Gyrate atrophy of the choroid and retina (GA) is an autosomal recessive chorioretinal degeneration caused by deficiency of the mitochondrial matrix enzyme, ornithine-delta-aminotransferase (OAT). To study the molecular basis of the mutations causing GA, we cloned and sequenced the human OAT cDNA and determined the intron-exon arrangement of the structural gene. Using the cDNA template, we synthesized antisense RNA probes and performed RNase A protection experiments with RNA from four Lebanese GA patients. We found a probe-target mismatch at the 5' end of the first coding exon and amplified this region of the patients' genomic DNA using the polymerase chain reaction. Sequence analysis showed a G----A transition, changing the initiator ATG (methionine) codon to ATA. This mutation segregates with the GA allele in both pedigrees. Initiation of translation at the closest in-frame methionine codon would truncate OAT by 138 amino acids, eliminating the entire mitochondrial leader sequence and 113 amino acids of the mature peptide.

Choroid↗
PubMed1980

Recognition of two initiation codons for the synthesis of phage fd gene 2 protein.

Bacteriophage fd gene 2 protein was specifically labeled with radioactive amino acids and was isolated from membranous cell structures as an apparently homogenous protein. Amino acid sequence analysis revealed that the protein was initiated at two distinct AUG codons close to the ribosome binding site. The two resulting translation products were found to begin with a deformylated methionine residue. Initiation at the first signal was used for 90% of the chains and at the second signal for 10% of the sequenced molecules. The use of one or the other chain start may influence functions of gene 2 protein.

Amino Acid Sequence↗
PubMed1987

The human glucocerebrosidase gene has two functional ATG initiator codons.

Gaucher disease is due to a deficiency in the activity of the enzyme glucocerebrosidase. Glucocerebrosidase is a lysosomal enzyme that presumably requires a signal peptide for transport across the membrane of the rough endoplasmic reticulum and glycosylation for transport into lysosomes. Human glucocerebrosidase cDNA contains two potential ATG start codons in its long open reading frame. The signal peptides that are initiated from each ATG are quite different in their hydrophobicity. We demonstrate that either ATG can function independently to produce active glucocerebrosidase enzyme in cultured fibroblasts. The glucocerebrosidase activity produced from translation products initiated at either ATG is found predominantly in the lysosomes.

Amino Acid Sequence↗
PubMed1994

The membrane-associated and secreted forms of the respiratory syncytial virus attachment glycoprotein G are synthesized from alternative initiation codons.

Respiratory syncytial (RS) virus synthesizes two mature forms of its attachment glycoprotein G: an anchored type II integral membrane form and a smaller form that is secreted into the medium. Here we demonstrate that these two forms are synthesized as distinct primary translation products of a single species of G protein mRNA by initiation at either of two different AUGs. Mutant cDNAs which eliminated one of the other of the two AUG codons near the 5' end of the G gene open reading frame were constructed. Analysis of the proteins synthesized from these cDNAs, either by translation of transcripts in a cell-free system or in cells infected with recombinant vaccinia viruses containing either one of the mutant cDNAs, showed that elimination of either the first or the second of these AUG codons abrogated the synthesis of the membrane-anchored or the secreted form of the protein, respectively. Additionally, two unglycosylated forms of G protein which comigrated with the unglycosylated G proteins expressed by these recombinant viruses were detected in RS virus-infected cells. Since the second AUG encodes a methionine residue that lies near the middle of the signal/anchor domain, initiation at this codon resulted in a protein with a hydrophobic amino terminus. This form of the glycoprotein was efficiently secreted from cells infected with the vaccinia virus recombinant, and the amino-terminal sequence of this protein was identical to that of G protein secreted from RS virus-infected cells. Our results demonstrate that the secreted form of RS virus G protein is produced by initiation at the second AUG codon of the G open reading frame, followed by proteolytic removal of the signal/anchor domain.

Amino Acid Sequence↗
PubMed1991

Two isoforms of murine hck, generated by utilization of alternative translational initiation codons, exhibit different patterns of subcellular localization.

Mammalian hck, a member of the src family of tyrosine kinases, is expressed predominantly in cells of the myeloid and B-lymphoid lineages. Using mutational analysis, we have investigated the molecular basis of two immunoreactive forms of murine hck of 56 and 59 kDa found in numerous hemopoietic cell types. Our results indicate that translation of murine p59hck initiates from a CTG codon located 21 codons 5' of an ATG that is utilized to generate p56hck. We provide evidence that two human hck isoforms are generated by the same mechanism. Subcellular fractionation studies reveal that while p59hck and p56hck are associated with membranes of various murine B-lymphoid and myeloid cell lines, p59hck alone is also located in the cytosol. In contrast to membrane-associated p59hck, which is metabolically labeled with [3H]myristic acid and exhibits amphiphilic properties in Triton X-114 detergent, cytosolic p59hck is hydrophilic, suggesting that it is not acylated. Possible mechanisms are proposed to account for these observations.

Amino Acid Sequence↗